Means and methods for increasing NK cells

Abstract

The present invention relates to (a) an antibody or antibody fragment that binds to an antigen expressed on the surface of target cells of NK cells, T cells and / or NKT cells, preferably on the surface of B cells or tumor cells; (b) an antibody or antibody fragment that binds to 4-1BB, NKG2D, NKp30, NKp46, NKp44, 2B4, CD28 or DNAM1; and (c) a fusion protein comprising IL-15, IL-2, IL-18, IL-21 or IL-12.

Description

[Technical Field]

[0001] The present invention relates to (a) an antibody or antibody fragment that binds to an antigen expressed on the surface of target cells of NK cells, T cells and / or NKT cells, preferably on the surface of B cells or tumor cells; (b) an antibody or antibody fragment that binds to 4-1BB, NKG2D, NKp30, NKp46, NKp44, CD28, DNAM1 or 2B4; and (c) a fusion protein comprising IL-15, IL-2, IL-21; IL-18 or IL-12.

[0002] This specification references several documents, including patent applications and manufacturers' manuals. While the disclosures of these documents are not considered relevant to the patentability of the present invention, they are incorporated herein by reference in their entirety. More specifically, all referenced documents are incorporated by reference to the same extent as if each individual document were specifically and individually indicated as being incorporated by reference. [Background technology]

[0003] Natural killer (NK) cells were first described in 1975, over 40 years ago. At that time, they were described, based on their function, as lymphocytes capable of recognizing and killing tumor cells without prior activation (Herberman, Nunn, Holden, & Lavrin, 1975; Kiessling, Klein, & Wigzell, 1975). Over the past 20 years, our understanding of the biology and function of NK cells has advanced significantly (Caligiuri, 2019). NK cells are described as large granular lymphocytes that make up about 10% of peripheral blood mononuclear cells (PBMCs). Due to their ability to detect and directly destroy malignant or infected cells, NK cells form a crucial part of the first line of defense of the innate immune system (J. Wagner, Pfannenstiel, & Waldmann, 2017). The cytotoxic response of NK cells to malignant or infected cells is direct and does not require prior antigen priming. Rather, the detection of malignant or infected cells is controlled by a set of activating and inhibitory receptors that recognize the absence of self proteins or the presence of stress ligands on target cells (PSABecker et al., 2016). The interaction between activators and inhibitors controls the response of NK cells, which in turn defines whether NK cells have been activated. When inhibitory receptors interact with HLA ligands, the receptors transmit a negative signal that leads to the suppression of the NK-mediated response. In contrast, when this type of interaction is absent, as can occur in virus-infected target cells or tumor cells, activating receptors transmit evoked signals to NK cells. This results in the killing of target cells (Bottino et al., 2003). The dominance of activating signals is another way in which NK cells induce the killing of target cells. NK cell suppression is substantially mediated by the CD94 / NKG2A heterodimer and polymorphic inhibitory killer cell immunoglobulin-like receptors (KIRs). The job of NK cells here is to distinguish between self and self-loss. This is important for preventing the killing of target cells in healthy self-tissue (Bottino et al., 2003).Both receptor types interact with the corresponding human leukocyte antigens (HLAs), which are molecules of the major histocompatibility complex (MHC) (PSABecker et al., 2016; Moretta et al., 1996; Perez-Villar et al., 1997). KIRs can be distinguished into KIR2D, KIR2DL1, KIR2DL2, KIR2DS4, KIR3DL1, etc. KIRs bind to HLA ligands by using the amino acid (AA) structure in the alpha-1 helix of the HLA molecule. HLA ligands are classified into three subgroups: HLA group 1 (C1), HLA group 2 (C2), and HLA-Bw4. All three subgroups bind to KIRs by a long extracellular immunoglobulin domain (PSABecker et al., 2016). Well-characterized activating receptors are the innate cytotoxic receptors (NCRs) NKp30 (CD337), NKp44 (CD336), and NKp46 (CD335), which recognize specific ligands on virus-infected or tumor cells. In addition to typical bacterial or viral structures such as hemagglutinin A (HA), NKp30 can also interact with a member of the B7 family of immune receptors called B7-H6 and an HLA-B related transcript called BAT-3 (Brandt et al., 2009; Strandmann et al., 2007). While NKp30 and NKp46 are present on all NK cells, NKp44 represents a receptor specifically expressed on activated NK cells (Vitale et al., 1998). In addition to NCRs, the C-type lectin-like receptor NKG2D (CD324) and DNAX accessory molecule-1 (DNAM-1; CD226) represent two other NK cell activating receptors (Andrade et al., 2015; PSABecker et al., 2016). The DNAM-1 molecule interacts with the poliovirus receptor (PVR; CD155) and the human plasma membrane glycoprotein nectin-2 (CD112) (Bottino et al., 2003).NKG2D interacts with unique long 16-binding proteins 1-6 (ULBP1-6) and MHC class I polypeptide-associated sequence A / B ligands (MIC A / B) (Lanier, 2015). Activation signaling occurs via transmembrane adapter proteins containing immunoreceptor tyrosine-based activation motifs (ITAMs). These ITAMs activate various signaling pathways upon phosphorylation. NKp30-related signals are transmitted via CD3ζ, while NKp46 signaling is transferred via CD3ζ chains and Fc receptor γ chains (FcRγ). NKp44 signaling is mediated by the 12kDa DNAX activating protein (DAP12), while NKG2D signaling is mediated via DAP10. The development and survival of NK cells in vivo and ex vivo requires cytokines such as IL-2, IL-4, IL-9, IL-15, IL-18, and IL-21 (Fujisaki et al., 2009; Miller et al., 2005). In addition to the described receptor molecules, NK cell activation can also be induced by these cytokines. Furthermore, IL-2 and IL-15 can be shown to have similar effects on NK cell development and survival (Giril et al., 1994). In addition, NK cells can be activated by the binding of IgG antibodies to the antigen CD16a (FcγRIIIa). Binding to CD16a can be shown to induce antibody-dependent cell-mediated cytotoxicity (ADCC), which leads to the killing of antibody-coated target cells mediated by NK cells (Bruenke et al., 2004). Phenotypically, NK cells are described as CD3- but CD56+ (Freud et al., 2005). Furthermore, human NK cells can be broadly classified into two subpopulations. Divide is the basis for CD56 cell surface expression. CD56dim NK cells, which exhibit low CD56 expression, and CD56bright NK cells, which exhibit increased CD56 expression, are distinguished. At the same time, CD56dim NK cells show higher levels of Fc receptor FcγRIIIa (CD16a) expression compared to CD56bright NK cells.These cells are CD16a-negative / low-expressing (Freud et al., 2005). Here, CD56dim NK cells show increased ADCC compared to CD56bright NK cells due to their CD16a expression. When NK cells are activated, immunosynapses / lytic synapses are formed. This formation can be divided into three steps: the initial step establishes the interaction between the NK cell and the target cell. In the effector step, the NK cell secretes vesicles containing lytic granules with granzyme and perforin into the intercellular interface and, consequently, into the target cell. Finally, there is a termination step in which the target cell is lysed. During the final step, perforin disrupts the target cell membrane, and granzyme lyses the target cell (Orange, 2008). In addition to perforin and granzyme, NK cells can also lyse target cells by binding to tumor necrosis factor-associated apoptosis-inducing ligand (TRAIL) (Smyth et al., 2005). Similarly, NK cells produce a variety of immunostimulatory cytokines and chemokines, such as tumor necrosis factor α (TNF-α) and interferon-γ (IFN-γ). In addition to immune surveillance, NK cells can interact with other immune cells via cytokines such as interleukin-3 (IL-3) or interleukin-10 (IL-10) (Vivier et al., 2011).

[0004] The foundation of NK cells in cancer treatment was laid by two studies in 1980 and 1984 that found that tumor cells could be effectively eliminated by NK cells in mice without prior stimulation (Mice et al., 1984; Riccardi, Santoni, & Barlozzari, 1980). NK cells are known to have the ability to recognize and destroy tumor cells through their MHC molecules. Just one year later, NK cells were demonstrated to spontaneously kill MHC class I-deficient tumor cells in vivo and in vitro (Ljunggren & Kaerre, 1985). While NK cell-based cancer immunotherapy is quite promising, tumors have been observed to employ various strategies to evade NK cell attack or to impair NK cell function and activity (Hu, Tian, ​​& Zhang, 2019). Even under normal conditions, NK cells constitute only a small fraction of peripheral blood mononuclear cells (PSABecker et al., 2016). Patients with various hematological malignancies and solid tumors may exhibit decreased NK cell counts and NK cell dysfunction (Fujisaki et al., 2009). Furthermore, decreased NK cell counts and NK cell dysfunction may be observed to correlate with poor prognosis in tumor patients. It has also been found that NK cells can exhibit an exhausted phenotype in tumor and chronic infection environments. Such phenotypes are typically characterized by decreased effector function and phenotypic alteration (Gardiner, 2017). Decreased effector function is associated with poor control of malignancies or infections due to reduced expression of cytolytic molecules such as granzymes and perforins. Phenotypic alteration is characterized by downregulation of specific activating receptors. NKG2D, CD16a, NKp30, NKp44, and NKp46, along with CD226, belong to the group of receptors that are typically reduced in tumor or chronic infection environments. Another phenotypic feature is upregulation of inhibitory receptors (Mamessier et al., 2011).

[0005] Therefore, since patients may benefit from a better ratio of effector cells to tumor cells, many NK cell-based therapeutic approaches focus on the proliferation and activation of fully functional NK cells and their adoption into the patient. Previous studies have shown that in vivo proliferation with cytokines is a promising approach. Increases in circulating NK cells and improved lytic function have been observed. Nevertheless, in vivo proliferation with cytokines has resulted in high toxic side effects (Burns et al., 2003). Furthermore, in vivo proliferation with cytokines (particularly IL-2) often also showed proliferation of regulatory T cells (Treg cells). These Treg cells can suppress NK cell activity (Gasteiger et al., 2013). To avoid these problems, a new approach has used ex vivo proliferation of NK cells. The advantage over in vivo proliferation is that patients are not expected to suffer from side effects. In addition, as mentioned above, under tumor conditions, NK cells exhibit an exhausted state characterized by NK cell dysfunction. When NK cells are grown in vivo in an immunosuppressive tumor environment, they tend to rapidly exhibit certain functional impairments (Fujisaki et al., 2009). Ex vivo growth offers the possibility of isolating NK cells from the immunosuppressive tumor environment and growing fully functional NK cells. There may be evidence that allogeneic NK cell infusion induces clinical remission in high-risk acute myeloid leukemia patients (Miller et al., 2005). Furthermore, patients with other cancer types may also be observed to benefit from NK cell infusion (Terme, Ullrich, Delahaye, Chaput, & Zitvogel, 2008).

[0006] Various ex vivo approaches are available and have been successful, but they are very costly in terms of technology or logistics. Cytokine-mediated ex vivo proliferation is one adduct. The development and survival of NK cells in vivo and ex vivo requires cytokines such as IL-2, IL-4, IL-9, IL-15, IL-18, and IL-21 (Fujisaki et al., 2009; Miller et al., 2005). Furthermore, heterodimeric cytokines called natural killer cell-stimulating factors (NKSFs) have been shown to promote NK cell-mediated cytotoxicity. Autologous NK cells can be observed to be proliferated and activated ex vivo. However, in contrast, ex vivo-proliferated NK cells can be shown to show no clinical response in cancer patients. Another approach is proliferation using a genetically modified K562 leukemia cell line. This modification involves the expression of membrane-bound interleukin (IL)-15 and 4-1BB ligand. The 4-1BB ligand specifically activates NK cells via binding to CD137 on NK cells. The use of K562 cells demonstrated high and specific proliferation of human NK cells and high cytotoxicity against tumor cells. However, the final cell product may contain traces of the genetically modified organism. Similarly, reinjected NK cell populations consistently contained tumor cells (Fujisaki et al., 2009). Various genetically modified feeder cells have been developed, but they generally exhibit the same limitations as described above. As an alternative, a bead-based approach has been described. Similar considerations apply to the ex vivo proliferation of T cells and / or NKT cells. [Overview of the Initiative] [Problems that the invention aims to solve]

[0007] Therefore, there is still a need for cost-effective and simple means and methods to enable the proliferation of NK cells, T cells, and / or NKT cells, particularly means and methods that eliminate the need for genetically modified feeder cells or microbeads. This need is addressed by this application. [Means for solving the problem]

[0008] Accordingly, the present invention relates in a first aspect to (a) an antibody or antibody fragment that binds to an antigen expressed on the surface of target cells of NK cells, T cells and / or NKT cells, preferably on the surface of B cells or tumor cells; (b) an antibody or antibody fragment that binds to 4-1BB, NKG2D, NKp30, NKp46, NKp44, CD28 DNAM1 or 2B4; and (c) a fusion protein comprising IL-15, IL-2, IL-21, IL-18 or IL-12.

[0009] The fusion protein of the present invention comprises three components according to (a), (b), and (c).

[0010] Components (a) and (b) are independently antibodies or antibody fragments. These two antibodies and / or antibody fragments bind to different antigens. The antibody or antibody fragment of (a) binds to antigens expressed on the surface of target cells of NK cells or T cells / NKT cells (preferably the surface of B cells or tumor cells), while the antibody or antibody fragment of (b) binds to 4-1BB, NKG2D, NKp30, NKp46, NKp44, CD28, 2B4, or DNAM-1.

[0011] The term "antibody" as used in accordance with the present invention includes, for example, polyclonal antibodies or monoclonal antibodies. Furthermore, it also includes fragments thereof that still retain the binding specificity required for the targets described herein. Antibody fragments include, in particular, Fab or Fab' fragments, Fd, F(ab')2, Fv or scFv fragments, single-domain VH or V-like domains, such as VhH or V-NAR domains, and multimer forms such as minibodies, diabodies, tribodies or triplebodies, tetrabodies, or chemically bound Fab' polymers (see, for example, Harlow and Lane, “Antibodies, A Laboratory Manual”, Cold Spring Harbor Laboratory Press, 198; Harlow and Lane, “Using Antibodies: A Laboratory Manual”, Cold Spring Harbor Laboratory Press, 1999; Altshuler EP, Serebryanaya DV, Katrukha AG. 2010, Biochemistry (Mosc)., vol. 75(13), 1584; Holliger P, Hudson PJ. 2005, Nat Biotechnol., vol. 23(9), 1126). Multimeric antibodies, in particular, include bispecific antibodies that can simultaneously bind to two different types of antigens. The first antigen can be found on the surface of target cells of NK cells, T cells, or NKT cells, preferably on the surface of B cells or tumor cells. The second antigen can be found in one of the proteins among 4-1BB, NKG2D, NKp30, NKp46, NKp44, CD28, or DNAM1 or 2B4. Non-limiting examples of bispecific antibody types include Biclonics (bispecific full-length human IgG antibodies), DART (biaffinity retargeting antibodies), and BiTE (consisting of two single-chain variable fragments (scFv) of different antibodies) molecules (Kontermann and Brinkmann (2015), Drug Discovery Today, 20(7):838-847).

[0012] The term "antibody" also includes embodiments such as chimeric (human constant domain, non-human variable domain), single-chain, humanized (human antibody except for non-human CDR) antibodies, and human antibodies.

[0013] Various techniques for producing antibodies are well known in the art and are described, for example, by Harlow and Lane (1988) and (1999) and Altshuler et al., 2010 (above). Therefore, polyclonal antibodies can be obtained from the blood of animals immunized with an antigen in a mixture with additives and adjuvants, and monoclonal antibodies can be produced by any technique that provides antibodies produced by serial cell line culture. Examples of such techniques are described, for example, in Harlow E and Lane D, Cold Spring Harbor Laboratory Press, 1988; Harlow E and Lane D, Using Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, 1999, and include hybridoma techniques, trioma techniques, human B-cell hybridoma techniques (see, e.g., Kozbor D, 1983, Immunology Today, vol. 4, 7; Li J, et al. 2006, PNAS, vol. 103(10), 3557), and EBV hybridoma techniques for producing human monoclonal antibodies (Cole et al., 1985, Alan R. Liss, Inc, 77-96). Furthermore, recombinant antibodies may be obtained from monoclonal antibodies or can be novelly prepared using various display methods such as phage, ribosome, mRNA, or cell display. A suitable system for the expression of recombinant (humanized) antibodies may be selected from, for example, bacteria, yeast, insects, mammalian cell lines, or transgenic animals or plants (see, for example, U.S. Patent No. 6,080,560; Holliger P, Hudson PJ. 2005, Nat Biotechnol., vol. 23(9), 11265). Furthermore, the techniques described for the production of single-chain antibodies (see, in particular, U.S. Patent No. 4,946,778) can be adapted to produce single-chain antibodies specific to the epitope defined in item (a) or (b) above.Surface plasmon resonance used in the BIAcore system can be used to enhance the efficiency of phage antibodies.

[0014] Since the constituent components (a) and (b) of the fusion protein are antibodies or antibody fragments that bind to separate antigens, it can also be said that the fusion protein of the present invention contains multispecific antibodies (note that the fusion protein may also contain additional antibodies or antibody fragments), particularly bispecific antibodies.

[0015] The term "multispecific antibody" used in accordance with the present invention includes binding motifs (e.g., antibodies and / or antibody fragments) that exhibit binding specificity to the targets defined in items (a) and (b) above. The multispecific antibody can also be extended by a third specificity that binds to a target on additional tumor cells or effector cells. The binding motifs of at least two different monoclonal antibodies can be included in the multispecific antibody in the form of full-length antibodies, but are also included in the term "antibody" as fragments thereof that still retain the binding specificity to the target, e.g., an antigen expressed on the surface of a tumor cell.

[0016] According to the present invention, polyspecific antibodies may have a multi-chain or single-chain form. The multi-chain or single-chain antibody form may be, for example, a minibody, diabody, vibody, tribody or triplebody, tetrabody, or a chemically bound Fab' polymer (see, for example, Harlow and Lane, “Antibodies, A Laboratory Manual”, Cold Spring Harbor Laboratory Press, 1988; Harlow and Lane, “Using Antibodies: A Laboratory Manual”, Cold Spring Harbor Laboratory Press, 1999; Altshuler EP, Serebryanaya DV, Katrukha AG. 2010, Biochemistry (Mosc)., vol. 75(13), 1584; Holliger P, Hudson PJ. 2005, Nat Biotechnol., vol. 23(9), 1126). Among these forms, the vibody form is preferred because the vibody form is described as an example. Vibody proteins, Fab-scFv fusion proteins, are created by adding an scFv fragment to the C-terminus of a Fab scaffold. In this type of polyspecific antibody, the bispecific fragment utilizes the in vivo innate heterodimerization of the Fd fragment (HC region of the Fab fragment) and the light chain. The heterodimerized scaffold can further incorporate additional functions such as scFv, scaffold proteins, and cytokines to form a bivalent bispecific molecule, or a trivalent bi or trispecific molecule. The vibody molecules Fab-L-scFv and Fab-H-scFv are bispecific and bivalent. This type has been shown to retain bispecific binding, low aggregation tendency, and stability under physiological conditions. Multi-chain types, in particular, include bispecific antibodies that can simultaneously bind to two different types of antigens.Non-limiting examples of bispecific antibody formats are Biclonics (bispecific full-length human IgG antibodies), DART (dual-affinity retargeting antibodies), and BiTE (composed of two single-chain variable fragments (scFv) of different antibodies) molecules (Kontermann and Brinkmann (2015), Drug Discovery Today, 20(7):838-847). Further bispecific antibody formats are discussed below herein.

[0017] As used herein, an antigen refers to a molecule or molecular structure that exists outside of a cell and can be specifically bound by an antibody or antibody fragment contained within the fusion protein of the present invention. An antigen contains an epitope (also referred to as an antigenic determinant), which is a part of the antigen recognized by the fusion protein of the present invention.

[0018] As described above, the antigen bound by the fusion protein of the present invention can be found on the surface of target cells of NK cells, T cells, and / or NKT cells (preferably, the surface of B cells or tumor cells) and within one of the proteins of 4-1BB, NKG2D, NKp30, NKp46, NKp44, 2B4, CD28, and DNAM-1.

[0019] As mentioned above, NK (natural killer) cells are a type of cytotoxic lymphocyte. NK cells are crucial to the innate immune system and belong to the rapidly expanding known family of innate lymphoid cells (ILCs), accounting for 5-20% of all circulating lymphocytes in humans. The role of NK cells is similar to that of cytotoxic T cells in the adaptive immune response of vertebrates. NK cells provide a rapid response to virus-infected cells and other intracellular pathogens, acting approximately 3 days after infection and responding to tumorigenesis. Normally, immune cells detect major histocompatibility complexes (MHC) presented on the surface of infected cells, triggering cytokine release and causing the death of infected cells through lysis or apoptosis. However, NK cells are unique because they have the ability to recognize and kill stressed cells even in the absence of antibodies and MHC, enabling a much faster immune response. NK cells are named "natural killers" because they do not require prior activation to kill cells lacking the "self" marker of MHC class I. This role is particularly important because harmful cells lacking the MHC I marker cannot be detected and destroyed by other immune cells such as T lymphocytes. NK cells are also important in the presence or absence of CD56 and CD3 (CD56 + CD3 - ) can be identified by this.

[0020] T cells, also known as T lymphocytes, are a type of white blood cell (leukocyte) that is an essential part of the immune system. T cells are one of two main types of lymphocytes that determine the specificity of the immune response to antigens (foreign substances) in the body (B cells are the second type). T cells originate in the bone marrow and mature in the thymus. In the thymus, T cells increase in number and differentiate into helper T cells, regulatory T cells, or cytotoxic T cells, or become memory T cells. They are then sent to peripheral tissues or circulate in the blood or lymphatic system. When stimulated by the appropriate antigen, helper T cells secrete chemical mediators called cytokines, which stimulate the differentiation of B cells into plasma cells (antibody-producing cells). Regulatory T cells work to control the immune response and are therefore called by that name. Cytotoxic T cells, activated by various cytokines, bind to and kill infected cells and cancer cells.

[0021] Natural killer T (NKT) cells are a heterogeneous group of T cells that share characteristics of both T cells and natural killer cells. Many of these cells recognize non-pleomorphic CD1d molecules. NKT cells make up only about 1% of all peripheral blood T cells and express various markers typical of NK cells, such as CD16 and CD56.

[0022] The target cells of NK cells, T cells, and / or NTK cells are not particularly limited. NK cells can target and kill abnormal cells, such as virus-infected cells and tumor-forming cells. Killing is mediated by cytotoxic molecules stored in secretory lysosomes, which are specialized extracellular organelles found in NK cells. Target cells may also be antigen-presenting cells (APCs), including dendritic cells (DCs). Similarly, target cells of T cells include cells infected with pathogens that replicate within the cell, tumor cells, and exogenous cells that enter the body as part of tissue transplantation.

[0023] The target cells are preferably B cells or tumor cells.

[0024] B cells (B lymphocytes) are a type of white blood cell subtype of lymphocyte. B cells function in the humoral immune portion of the adaptive immune system. B cells produce antibody molecules, which can be secreted or inserted into the plasma membrane and function as part of the B cell receptor. Naive or memory B cells, when activated by an antigen, proliferate and differentiate into effector cells known as plasmablasts or plasma cells, which secrete antibodies. Furthermore, B cells present antigens (B cells are also classified as professional antigen-presenting cells (APCs)) and secrete cytokines. Unlike the other two types of lymphocytes, T cells and natural killer cells, B cells express the B cell receptor (BCR) on their cell membrane. The BCR allows B cells to bind to foreign antigens and initiate an antibody response against them.

[0025] Tumor cells are abnormal cells that differ in many ways from normal somatic cells. Normal cells become tumor cells when a series of mutations causes them to grow and divide uncontrollably. Also, unlike normal cells, tumor cells may have the ability to invade nearby tissues and / or spread to distant areas of the body. As a result of the series of mutations, antigens not expressed on normal cells are often expressed on the surface of tumor cells. In addition, tumor cells express a variety of antigens also found in healthy tissues. However, assuming that the expression pattern is limited and / or that the expression is restricted to specific tissues or cell types in healthy tissues, such antigens can also be used as target structures. Such antigens are referred to herein as antigens expressed on the surface of tumor cells. Antigens are preferably not expressed on normal cells. Therefore, “bispecific conjugated antibodies” can bind specifically or at least very preferentially to tumor cells. The tumors referred to herein may be malignant or benign tumors. The tumors are preferably malignant tumors, also referred to herein as cancers.

[0026] As will be explained in more detail below, all of the proteins 4-1BB, NKG2D, NKp30, NKp46, NKp44, 2B4, CD28, or DNAM-1 are expressed on the surface of NK cells, T cells, and / or NKT cells, and all of them are involved in the activation of these cells.

[0027] 4-1BB(ILA / CD137) is a member of the tumor necrosis factor receptor family expressed on activated T lymphocytes and NK cells.

[0028] NKG2D is a transmembrane protein belonging to the NKG2 family of C-type lectin-like receptors. NKG2D plays a crucial role in immune surveillance of tumors and pathogens. In humans, NKG2D is expressed by NK cells and cytotoxic thymocytes and recognizes "induced self proteins" that are often expressed on the cell surface after viral infection or malignant transformation. Human NKG2D ligands include MHC class I-associated chains (MICs) A and B, as well as UL16-binding proteins (ULBPs) 1-6. Recognition of these danger signaling antigens leads to cell activation via an intracellular activation pathway by the NKG2D-associated adapter protein, 10kDa DNAX-activating protein (DAP10). In NK cells, this signaling promotes innate cytotoxicity.

[0029] NKp30 (CD337) is a stimulatory receptor on human NK cells involved in tumor immunity, and can promote or terminate the maturation of dendritic cells.

[0030] NKp46 is a major NK cell activating receptor involved in the removal of target cells being killed by NK cells.

[0031] NKp44 (CD336) is a member of the innate cytotoxic receptor (NCR). It is an activating receptor and plays a crucial role in most functions expressed by activated NK cells and other NKp44+ immune cells.

[0032] DNAM-1 (CD226) is an activating receptor belonging to the Ig superfamily and is constitutively expressed by most NK cells, T cells, macrophages, and dendritic cells (DCs). DNAM-1 interacts with LFA-1, which is required for its functional activity in both NK cells and cytotoxic T cells. Ligands for DNAM-1 (DNAM1L) include nectin-2 / CD112 and PVR / CD155, which belong to the nectin / nectin-like family of adhesion molecules. The activating effect of DNAM-1 can be counteracted by TIGIT. TIGIT is a recently identified inhibitory receptor that binds to PVR and is expressed by T cells and NK cells.

[0033] 2B4 (CD244) is a protein expressed on NK cells and some T cells. The interaction between NK cells and target cells via this receptor is thought to regulate the cytolytic activity of NK cells.

[0034] CD28 is a protein expressed on T cells that provides a co-stimulatory signal necessary for T cell activation and survival.

[0035] Since the fusion protein of the present invention must be suitable for proliferating NK cells, particularly NK cells with high ADCC activity, it is noteworthy that antibodies or antibody fragments that bind to CD16 are unsuitable as the second component (b) of the fusion protein of the present invention. CD16a (FcγRIIIa) is considered to be the most potent cytotoxic trigger in NK cells. While high-affinity binding of CD16a may be desirable from the viewpoint of inducing potent cytotoxicity against target cells, the high-affinity CD16a binding domain is undesirable in relation to the fusion protein of the present invention. This is because it is well established that crosslinking of CD16a with antibodies downregulates CD16a expression. This downregulation is expected to result in the preparation of NK cells that are impaired in terms of ADCC induction. In contrast to natural IgG, the dramatic effect of CD16 antibody binding to CD16a is described, for example, in Romee et al. (2013), Blood, (18):3599-608, and Capuano et al. (2017), Oncoimmunology, 6(3):e1290037.

[0036] As a third component (c), the fusion protein of the present invention comprises an interleukin selected from the group consisting of IL-15, IL-2, IL-18, IL-21, or IL-12. Of this list, IL-15 is most preferred.

[0037] A third component (c) of the fusion protein of the present invention is also intended herein to be a variant, cleaved form, or variant of an interleukin selected from the group consisting of IL-15, IL-2, IL-18, IL-21, or IL-12. The main functions of interleukins are to regulate cell growth, cell differentiation, and activation during inflammatory and immune responses, and one or more of these functions are preferably retained in a variant, cleaved form, or variant of an interleukin selected from the group consisting of IL-15, IL-2, IL-18, IL-21, or IL-12.

[0038] Interleukins (ILs) are a group of cytokines (secreted proteins and signaling molecules) expressed and secreted by leukocytes and several other somatic cells. The human genome codes for more than 50 interleukins and related proteins.

[0039] IL-15, IL-2, IL-21, IL-18, or IL-12 are all so-called pro-inflammatory cytokines. Pro-inflammatory cytokines are mainly produced by activated macrophages and are involved in the upregulation of inflammatory responses. NK cells respond to specific cytokines, and their activity can be enhanced by stimulation with IL-15, IL-2, IL-18, IL-21, or IL-12 (de Rham et al. (2007), Arthritis Res Ther; 9(6):R125 and Rezani and Rouce (2015), Front.Immunol.).

[0040] Interleukin-15 (IL-15) is a cytokine structurally similar to interleukin-2 (IL-2). Like IL-2, IL-15 binds to a complex composed of the IL-2 / IL-15 receptor beta chain (CD122) and common gamma chain (gamma-C, CD132), and transmits signals through this complex. IL-15 is secreted by mononuclear phagocytes (and some other cells) after viral infection. This cytokine induces the proliferation of natural killer cells, which are cells of the innate immune system whose primary role is to kill virus-infected cells. IL-15R is expressed by NK cells.

[0041] Interleukin-2 (IL-2) is an interleukin, a type of cytokine signaling molecule in the immune system. It is a 15.5-16 kDa protein that regulates the activity of leukocytes (white blood cells, mostly lymphocytes) that are responsible for immunity. IL-2 is part of the body's natural response to microbial infection and is involved in distinguishing between foreign ("non-self") and "self." IL-2 mediates its effects by binding to IL-2 receptors expressed by lymphocytes. The main sources of IL-2 are activated CD4+ T cells and activated CD8+ T cells. Dimeric IL-2R is expressed by memory CD8+ T cells and NK cells, while regulatory T cells and activated T cells express high levels of trimer IL-2R.

[0042] Interleukin-18 (IL-18), also known as interferon-gamma inducer, is a cytokine. IL-18 is a pro-inflammatory cytokine. Many cell types, both hematopoietic and non-hematopoietic cells, have the potential to produce IL-18. IL-18 production was first recognized in Kupffer cells, which are hepatic macrophages. However, IL-18 is constitutively expressed in non-hematopoietic cells such as intestinal epithelial cells, keratinocytes, and endothelial cells. IL-18 can modulate both innate and adaptive immunity, and dysregulation of this immunity can lead to autoimmune or inflammatory diseases.

[0043] Interleukin-21 (IL-21) is a cytokine with potent regulatory effects on immune system cells, including natural killer (NK) cells and cytotoxic T cells, which can destroy virus-infected or cancerous cells. This cytokine induces cell division / proliferation in its target cells. The IL-21 receptor (IL-21R) is expressed on the surface of T cells, B cells, and NK cells.

[0044] Interleukin-12 (IL-12) is an interleukin spontaneously produced by dendritic cells, macrophages, neutrophils, and human B lymphoblastoid cells (NC-37) in response to antigen stimulation. IL-12 plays a crucial role in the activity of natural killer cells and T lymphocytes. IL-12 mediates the enhancement of cytotoxic activity in NK cells and CD8+ cytotoxic T lymphocytes. A link also appears between IL-2 and IL-12 signaling in NK cells. IL-2 stimulates the expression of two IL-12 receptors, IL-12R-β1 and IL-12R-β2, maintaining the expression of key proteins involved in IL-12 signaling in NK cells. Enhanced functional response is demonstrated by IFN-γ production and target cell killing. IL-12R is expressed by NK cells.

[0045] Therefore, it should be noted that the order of components (a), (b), and (c), and the method of binding the components are not particularly limited, as long as the two antibodies or antibody fragments of components (a) and (b) can bind to the antigen and component (c) can bind to the interleukin receptor on NK cells and their target cells. The order of the components may be, for example, (a)(b)(c) from the N-terminus to the C-terminus, but may also be (c)(b)(a), (c)(a)(b), (b)(a)(c), (b)(c)(a), or (a)(c)(b).

[0046] The components are preferably linked via a linker, which is preferably a mobile peptide linker, as further described below herein.

[0047] As can be seen from the above description, the fusion protein of the present invention binds to target cells via its antibody component (a) and provides transpresentation of IL-15, IL-2, IL-18, IL-21, or IL-12 of component c) of the fusion protein to bind to the respective interleukin receptors on the surface of NK cells, T cells, and / or NTK cells. This then likely leads to upregulation of the target antigen of component (b), further linking NK cells, T cells, and / or NTK cells to their target cells (see Figure 1B). The simultaneous binding of 4-1BB, NKG2D, NKp30, NKp46, NKp44, 2B4, CD28, or DNAM-1 by component (b) of the fusion protein to IL-15R, IL-2R, IL-18R, IL-21R, or IL-12R on the surface of NK cells by component (c) triggers two signaling cascades that activate NK cells, T cells, and / or NTK cells, thereby leading to their proliferation. The binding of NK cells, T cells, and / or NTK cells to their target cells likely further improves the proliferation of NK cells, T cells, and / or NTK cells by providing overcrosslinking of components b) and c), thereby enhancing cytokine transpresentation, considering a positive feedback loop between the target cells and the NK cells, T cells, and / or NTK cells. Furthermore, the close proximity of NK cells, T cells, and / or NTK cells to their target cells enhances the killing efficacy of NK cells, T cells, and / or NTK cells against malignant target cells, but not to the same extent for non-malignant cells.

[0048] As shown in the following appendix examples herein, an exemplary fusion protein of the present invention (RTX-CD137scFv-IL-15 / RTX-DuoFab-CD137scFv-IL-15), comprising one or two (DuoFab)CD20-directed Fab fragments as component (a), the agonist anti-4-1BB(CD137)scFv as component (b), and the sushi domain of the IL-15 receptor fused to human IL-15 as component (c), induces potent NK cell proliferation upon binding to NK target cells (exemplary autologous B cells). This is thought to be because the fusion protein binds to IL-15R and 4-1BB on the surface of NK cells. The same essentially applies to further exemplary fusion proteins of the present invention comprising (a) one or two (DuoFab)BCMA-targeted Fab fragments, the agonist anti-4-1BB(CD137)scFv, and the sushi domain of the IL-15 receptor fused to human IL-15 (BCMA-CD137scFv-IL-15 / BCMA-DuoFab-CD137scFv-IL-15), (b) CD19-CD137scFv-IL-15, (c) RTX-CD137scFvdss-IL-15, (d) RTX-CD137scFv-IL-2, (e) RTX-NKp46scFv-IL-15, and (f) RTX-NKG2DscFv-IL-15. Data for CD20, BCMA, and CD19 as component (a), CD137 (optionally with added disulfide bonds), NKp46, and NKG2D as component (b), and IL-15 (with a sushi domain) and IL-2 as component (c) indicate that all three components can be altered. Potent NK cell proliferation was achieved with all of these exemplary fusion proteins. In particular, the NK cell proliferation and cytolytic activity obtained by the exemplary fusion proteins was significantly potent compared to the prior art molecular designs (SEQ ID NOs: 79 and 80), as described in Kerner, Mol Cancer Ther, 2014; Beha, Mol Cancer Ther, 2019. This prior art molecular design is based on a 4-1BB ligand to stimulate CD137 instead of using CD137scFv.Data from one or two Fab fragments demonstrate that the specific antibody type used in the fusion protein of the present invention is not limited. No significant differences were observed between one Fab fragment and two Fab fragments in terms of NK cell proliferation or the cytotoxic activity of the proliferated NK cells.

[0049] The following examples in this specification demonstrate that all three components (a), (b), and (c) of the fusion protein of the present invention are necessary for complete activity. The proliferation rate ranged from 10-fold to 10,000-fold. The proliferated NK cells showed high cytotoxicity against a wide range of tumor cell lines representing various tumor entities. Importantly, the proliferated, highly activated NK cells did not attack non-malignant B cells, indicating that NK cells proliferated by the fusion protein of the present invention remain physiologically regulated. The cytotoxic activity of the proliferated NK cells can be further enhanced by combining them with therapeutic antibodies, such as bispecific therapeutic antibodies. The fusion protein of the present invention can also be used to highly proliferate NK cells from multiple myeloma patients.

[0050] According to a preferred embodiment of the first aspect of the present invention, the antigen of (a) is selected from the group consisting of CD20, BCMA, CD19, CD22, CD37, CD38, CD7, CD33, CD44, CD54, CD64, CD75s, CD79b, CD96, CD123, CD317, CD319, FCRL5, EGFR, B7-H3, HER2, EpCAM, CEA, GD2 and claudin 6 / 18, Trop-2, ROR1, PSMA, FolR1, STEAP1, Her3, uPAR, Muc-1, cMet, CXCR4, SAP-1, Muc-16, TAG-72, HLA-DR, CD30, DLL4, CD221, mesothelin, GPRC5D, nectin-4, LIV-1 and tissue factor, and is preferably CD20 or BCMA.

[0051] The fusion protein of the present invention may contain binding domains for two or more of these antigens. Therefore, the fusion protein of the present invention can bind to two or more antigens selected from the group consisting of CD20, BCMA, CD19, CD22, CD37, CD38, CD7, CD33, CD44, CD54, CD64, CD75s, CD79b, CD96, CD123, CD138, CD317, CD319, FCRL5, EGFR, B7-H3, HER2, EpCAM CEA, Claudin 6 / 18, ROR1, PSMA, FolR1, STEAP1, Her3, uPAR, Muc-1, cMet, CXCR4, SAP-1, Muc-16, TAG-72, HLA-DR, CD30, DLL4, CD221, Mesothelin, GPRC5D, Nectin-4, LIV-1, and tissue factor, wherein the two or more antigens preferably include CD20 and / or BCMA. The expression of all these antigens is known to be involved in cancer development, and all of these antigens can be found in certain cancer cells.

[0052] The B lymphocyte antigen CD20, or CD20, is expressed on the surface of all B cells, starting in the pro-B stage (CD45R+, CD117+) and gradually increasing in concentration until maturation. CD20 has been found in B-cell lymphoma, hairy cell leukemia, chronic lymphocytic leukemia (CLL), B-cell acute lymphoblastic leukemia (ALL), and melanoma cancer stem cells.

[0053] B-cell maturation antigen (BCMA), also known as tumor necrosis factor receptor superfamily member 17 (TNFRSF17), is a protein encoded in humans by the TNFRSF17 gene. TNFRSF17 is a cell surface receptor of the TNF receptor superfamily that recognizes B-cell activator (BAFF). BCMA recognizes B-cell activator (BAFF). BCMA expression is associated with leukemia, lymphoma, and multiple myeloma.

[0054] CD19 is a transmembrane protein expressed in B cells. As a marker for B cells, CD19 is used to diagnose and target cancers arising from this type of cell, particularly B-cell lymphoma, acute lymphoblastic leukemia (ALL), and chronic lymphocytic leukemia (CLL).

[0055] CD22 is a molecule belonging to the SIGLEC family of lectins. It is found on the surface of mature B cells, and to a lesser extent, also in some immature B cells. CD22 is also used to diagnose and target B cell-derived cancers, such as acute lymphoblastic leukemia (ALL).

[0056] CD37 is a member of the transmembrane 4 superfamily. CD37 expression is limited to immune system cells, with the highest abundance in mature B cells and lower expression in T cells and myeloid cells. In cancer, CD37 is highly expressed on malignant B cells in various B-cell lymphomas and leukemias, including non-Hodgkin lymphoma (NHL) and CLL.

[0057] CD38 is a glycoprotein found on the surface of many immune cells (white blood cells), including CD4+, CD8+, B lymphocytes, and natural killer cells. CD38 is also expressed in various hematological malignancies, including NHL, MM, CLL, and ALL.

[0058] CD7 encodes a transmembrane protein that is a member of the immunoglobulin superfamily. CD7 is found in thymocytes and mature T cells. CD7 is expressed in T-cell leukemias and lymphomas and is a prognostic marker for leukemia.

[0059] CD33 is a transmembrane receptor expressed on myeloid cells. It is a target used in the treatment of patients with acute myeloid leukemia.

[0060] CD44 is a cell surface glycoprotein involved in cell-cell interactions, cell adhesion, and migration. CD44 is expressed in numerous mammalian cell types. CD44 diversity has been reported as a cell surface marker for some breast and prostate cancer stem cells.

[0061] CD54 is a cell surface glycoprotein normally expressed on endothelial cells and immune system cells. CD54 plays an important role in ocular allergies, replenishing pro-inflammatory lymphocytes and mast cells to promote type I hypersensitivity reactions.

[0062] CD64 is a type of membrane-bound glycoprotein known as an Fc receptor that binds with high affinity to monomeric IgG antibodies. CD64 is found in macrophages and monocytes. Neutrophil CD64 expression is increased in inflammatory autoimmune diseases.

[0063] CD75s are alpha-2,6-sialylated carbohydrate epitopes expressed by mature B cells (particularly germinal center B cells), erythrocytes, and some epithelial cells. CD75s have been identified as a promising target for immunotherapy of mature B cell malignancies.

[0064] CD79b is a B-cell antigen receptor complex-associated protein beta chain. Diseases associated with CD79b include autosomal recessive agammaglobulinemia6 and non-Bruton agammaglobulinemia.

[0065] CD96 is a transmembrane glycoprotein with three extracellular immunoglobulin-like domains that is expressed by resting NK cells. CD96 has been reported to correlate with the immune profile and clinical outcomes of gliomas.

[0066] CD123 is a molecule found in cells that assist in the signaling of interleukin-3, an important soluble cytokine in the immune system, such as pluripotent progenitor cells of hematopoietic cells. CD123 is expressed across all subtypes of acute myeloid leukemia (AML), including leukemia stem cells.

[0067] CD138 (or syndecan 1) is a protein encoded in humans by the SDC1 gene. This protein is a transmembrane (type I) heparan sulfate proteoglycan. CD138 functions as an endogenous membrane protein and is involved in cell proliferation, cell migration, and cell-matrix interactions via its receptors for extracellular matrix proteins. CD138 is a sponge for growth factors and chemokines, binding primarily via heparan sulfate chains.

[0068] CD317 is a lipid raft-related protein expressed in mature B cells, plasma cells, and plasmacytoid dendritic cells, as well as many other cells. It is expressed only in response to stimulation from the IFN pathway. Several reports describe the expression of CD317 in various types of malignancies, including lung cancer, leukemia, and lymphoma.

[0069] CD319 (also known as CS1 (CD2 subset-1), CRACC, and SLAMF7) is a single-pass type I transmembrane glycoprotein expressed on NK cells, a subset of mature dendritic cells, activated B cells, and cytotoxic lymphocytes, but not on promyelocytic B cell lines or T cell lines. CD319 is a robust marker for normal and malignant plasma cells in multiple myeloma.

[0070] FCRL5 (Fc receptor-like protein 5, also known as CD307) is a receptor that recognizes intact IgG, and presumably enables B cells to sense the quality of Ig. Diseases associated with FCRL5 include hairy cell leukemia and lymphoma.

[0071] EGFR (epidermal growth factor receptor) is a transmembrane protein that is a receptor for members of the epidermal growth factor family (EGF family), which are extracellular protein ligands. In many cancer types, cancer can develop as a result of mutations that affect EGFR expression or activity.

[0072] B7-H3 (or CD276) is a 316-amino acid type I transmembrane protein that exists in two isoforms determined by its extracellular domain. In mice, the extracellular domain consists of a single pair of immunoglobulin variable region (IgV)-like and immunoglobulin constant region (IgC)-like domains, while in humans, due to exon duplication, it consists of either one pair (2Ig-B7-H3) or two identical pairs (4Ig-B7-H3). In non-malignant tissues, B7-H3 plays a primarily suppressive role in adaptive immunity, inhibiting T cell activation and proliferation.

[0073] HER2 (receptor tyrosine protein kinase erbB-2, also known as CD340) is a receptor that plays a crucial role in normal cell growth and differentiation. Overexpression of HER2 is known to occur in, for example, breast cancer, ovarian cancer, gastric cancer, lung adenocarcinoma, and uterine cancer.

[0074] EpCAM (epidermal cell adhesion molecule) is a transmembrane glycoprotein that mediates Ca2+-independent isoplastic cell adhesion in epithelium. EpCAM is overexpressed in many cancers and cancer stem cells, making it an attractive target for immunotherapy.

[0075] CEA (carcinoembryonic antigen) describes a series of highly related glycoproteins involved in cell adhesion. CEA is normally produced in gastrointestinal tissue during fetal development, but production ceases before birth. In adults, CEA is primarily expressed in (malignant and benign) tumor cells.

[0076] GD2 is a disialogangliside that is only expressed to a limited extent in healthy tissues. In certain tumors, GD2 is widely expressed and associated with cancer development. This antigen is a target in the treatment of neuroblastoma.

[0077] CLDN (claudin), along with occludins, refers to members of the protein family that are the most important components of tight junctions (zonulae occludentes). Alterations in the expression of several claudin proteins, particularly claudin-1, -3, -4, and -7, have been associated with the development of various cancers. Furthermore, CLDN6 and CLDN18.2 are attractive targets for immunotherapy.

[0078] Tumor-associated calcium signaling transducer 2 is also known as Trop-2 and epithelial glycoprotein-1 antigen (EGP-1). Trop-2 is a cancer-associated antigen defined by the monoclonal antibody GA733. This antigen is a member of a family that includes at least two type I membrane proteins. It transmits intracellular calcium signals and functions as a cell surface receptor.

[0079] The tyrosine protein kinase transmembrane receptor ROR1, also known as neurotrophic tyrosine kinase receptor-associated 1 (NTRKR1), is an enzyme. ROR1 is a member of the receptor tyrosine kinase-like orphan receptor (ROR) family. This protein regulates neurite growth in the central nervous system.

[0080] PSMA (prostate-specific membrane antigen) is a membrane protein highly expressed in prostate adenocarcinoma, while its expression is limited in benign and extraprostatic tissues. Therefore, it represents a promising target in prostate cancer.

[0081] Folate receptor 1 (FOLR1) is a transmembrane protein that is overexpressed in selected solid tumors, for example, in more than one-third of gastric cancer patients. It is hardly expressed in normal tissues.

[0082] STEAP1 (six-transmembrane prostatic antigen 1) is expressed in approximately 90% of prostate cancers and is also expressed in other malignant tumors. STEAP1 is associated with tumor invasiveness and progression and is expressed only at low levels in normal tissue.

[0083] Her3 is a heterodimer partner of other EGFR family members and may regulate resistance via EGFR / HER2. Upregulation of HER3 is associated with several malignancies and promotes tumor progression through interactions with other receptor tyrosine kinases.

[0084] uPAR (urokinase-type plasminogen-activating factor receptor, CD87) belongs to the lymphoid antigen 6 superfamily. The uPAR receptor is a single-chain membrane glycoprotein receptor that is fixed to the cell membrane by glycosylphosphatidylinositol (GPI) binding. It is expressed at low levels in healthy tissues and at high levels in malignant tumors. uPAR is highly expressed in solid tumor tissues such as the breast, lung, ovaries, and prostate, as well as in several other entities, including some hematological malignancies.

[0085] Muc-1 is specifically overexpressed and abnormally glycosylated in many types of cancer, such as gastrointestinal cancer.

[0086] cMet is abnormally expressed in various malignancies, particularly non-small cell lung cancer, gastrointestinal cancer, and hepatocellular carcinoma.

[0087] Upregulation of CXCR4 (CXC-motif chemokine receptor 4) in various cancer entities is widely recognized, and this receptor is a suitable target for solid tumors, including adrenocortical carcinoma and small cell lung cancer.

[0088] SAP-1 (gastric cancer-associated protein tyrosine phosphatase 1) is a human transmembrane protein tyrosine phosphatase. SAP-1 is expressed in large quantities in colon and pancreatic cancer cells.

[0089] Abnormal overexpression of Muc-16 (CA125) has been observed in several malignancies, including ovarian cancer, pancreatic cancer, breast cancer, and lung cancer. Due to its abnormal overexpression, Muc-16 has emerged as a potential target in immunotherapy.

[0090] TAG-72 (tumor-associated glycoprotein 72 antigen) is found at high levels on the surface of several cancer types, including ovarian cancer.

[0091] HLA-DR (Human Leukocyte Antigen-DR) is one of the three polymorphic isotypes of the class II major histocompatibility complex antigen. Because HLA-DR is expressed at high levels in various hematological malignancies, it is an interesting target for antibody-based lymphoma therapy.

[0092] CD30 is a member of the tumor necrosis factor receptor superfamily and is expressed in certain hematopoietic malignancies, including cutaneous T-cell lymphoma, anaplastic large cell lymphoma, and Hodgkin lymphoma. It is an established target for antibody-based immunotherapy (e.g., brentuximab-vedotin).

[0093] DLL4 (Delta-like canonical Notch ligand 4). The delta gene family encodes Notch ligands characterized by a DSL domain, EGF repeat, and transmembrane domain. DLL4 is associated with gastric cancer.

[0094] CD221 (IGF-1R) is an important tyrosine kinase receptor that plays a crucial role in mitosis induction, angiogenesis, transformation, apoptosis, and cell motility. Various preclinical and epidemiological studies have identified the role of IGF-1R in carcinogenesis, including prostate cancer, breast cancer, colorectal cancer, and lung cancer.

[0095] Mesothelin expression has been detected in many solid tumors, including ovarian cancer, pancreatic adenocarcinoma, lung and uterine malignancies, and cholangiocarcinoma. More recently, mesothelin has also been discussed as a therapeutic target in AML.

[0096] GPRC5D (G protein-coupled receptor, class C, group 5, member D) is a member of the G protein-coupled receptor (GPCR) family and is a therapeutic target in multiple myeloma.

[0097] Nectin cell adhesion protein 4 (nectin-4) is overexpressed in various human malignancies, and its abnormal expression is correlated with cancer progression. Nectin-4 is overexpressed in urothelial carcinoma and several other malignancies.

[0098] LIV-1 is a member of the solute carrier family 39, a multispan transmembrane protein with metalloproteinase activity. It functions as a therapeutic target for the treatment of metastatic breast cancer.

[0099] Tissue factor is expressed by various cancers to serve as a target for antibody-based therapeutic approaches.

[0100] According to a preferred embodiment of the first aspect of the present invention, each of the antibody fragments (a) and (b) is independently selected from Fab, scFv, Fv, VHH, and dAb, wherein the antibody fragment (a) is preferably Fab and the antibody fragment (b) is preferably scFv.

[0101] The specific type of fusion protein of the present invention is not particularly limited, but a fusion protein according to this preferred embodiment comprises, independently, Fab, scFv, Fv, VHH, or dAb as antibody fragments (a) and (b).

[0102] Fab (antigen-binding fragment), scFv (single-chain variable fragment), Fv (variable fragment), VHH (variable domain of heavy-chain-only antibody), and dAb (domain antibody) are well-known fragments of full-length (or complete) antibodies. A full-length (or complete) antibody consists of two copies each of the entire immunoglobulin light chain and heavy chain. Among this list of antibody fragments, the scFv fragment is particularly preferred for inclusion in the fusion protein of the present invention.

[0103] Characteristic properties of antibody fragments compared to full-length antibodies include, for example, smaller size, monovalent antigen binding, absence of FcR binding, generally lack of complex glycosylation, and / or robust biophysical properties.

[0104] The type of fusion protein of the present invention preferably comprises IgG and scFv or Fab and scFv as components (a) and (b), more preferably comprising an IgG-scFv or Fab-scFv fusion protein, or vice versa. In the first case, IgG (i.e., a whole IgG antibody) is fused to the scFv fragment, and in the second case, the Fab fragment is fused to the scFv fragment.

[0105] For example, the fusion protein of the present invention may include an scFv fragment, which is preferably fused via a mobile peptide linker or a preferred embodiment thereof as described below herein.

[0106] According to a preferred option of the above embodiment of the first aspect of the present invention, the Fab scaffold specifically binds to antigen (a), and the scFv fragment specifically binds to antigen (b).

[0107] A Fab scaffold may be either a single Fab fragment or two Fab fragments (also referred to herein as DuoFab). This particular Fab-scFv type contained in the fusion protein of the present invention is described in the examples of this application at the time of filing (single Fab and DuoFab) and is therefore particularly preferred. Fab-scFv types having an intermediate molecular mass of about 75 kDa, in contrast to tandem scFv types, may not be removed by renal clearance, thereby having a longer in vivo half-life. Compared to types such as IgG, their smaller size exhibits favorable properties when mediating synapse formation between target cells and effector cells. Eliminating the need for the use of multiple scFv fragments reduces the tendency of such Fab-scFv molecules to form multimers or aggregates.

[0108] If an even longer in vivo half-life is desired, the Fab-scFv type can further incorporate an Fc domain. Such molecules, with a molecular weight of approximately 125 kDa, are even smaller than typical IgG antibodies and therefore can demonstrate favorable properties in terms of tissue permeability.

[0109] In this regard, it should be understood that while Fc scaffolds do not contain antigen-binding sites, they are further components of the fusion. Fc scaffolds can, for example, increase the in vivo serum stability and retention time of multispecific antibodies.

[0110] Furthermore, for in vivo applications, the Fab-scFv type may, if desired, be a DuoFab-scFv type. Such molecules exhibit increased retention time (see Example 3) and are advantageous for in vivo applications.

[0111] As discussed above in this specification, exemplary fusion proteins of the present invention include, as component (a), one or two (DuoFab)CD20-directed Fab fragments (hereinafter also referred to as RTX = rituximab), one or two (DuoFab)BCMA-directed Fab fragments (hereinafter also referred to as BCMA), or a CD19-directed Fab fragment.

[0112] The DNA and protein sequences of the RTX light chain are sequence numbers 1 and 2, and the DNA and protein sequences of the RTX heavy chain VH are sequence numbers 3 and 4.

[0113] Therefore, it is preferable that component (a) contains sequences that are at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, and 100% (in increasing order of priority) identical to sequence numbers 2 and / or 4, or that it is coded by sequences that are at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, and 100% (in increasing order of priority) identical to sequence numbers 1 and / or 3.

[0114] The DNA and protein sequences of the BCMA light chain are sequence numbers 5 and 6, and the DNA and protein sequences of the BCMA heavy chain VH are sequence numbers 7 and 8.

[0115] Therefore, component (a) may also be coded by sequences that are at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, and 100% identical (in increasing order of priority) to sequence numbers 6 and / or 8, or by sequences that are at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, and 100% identical (in increasing order of priority) to sequence numbers 5 and / or 7.

[0116] The light chain DNA and protein sequences of the CD19-directed Fab fragment are sequence numbers 47 and 48, and the heavy chain VH DNA and protein sequences of the CD19-directed Fab fragment are sequence numbers 49 and 50.

[0117] Therefore, it is preferable that component (a) contains sequences that are at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, and 100% (in increasing order of priority) identical to sequence numbers 48 and / or 50, or that it is coded by sequences that are at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, and 100% (in increasing order of priority) identical to sequence numbers 47 and / or 49.

[0118] As discussed above in this specification, exemplary fusion proteins of the present invention include, as component (b), the agonist anti-4-1BB(CD137)scFv (also referred to herein as CD137), the agonist NKp46scFv, the agonist NKG2DscFv, or the agonist anti-4-1BB(CD137)scFvdss (having an additional disulfide bond), with the agonist anti-4-1BB(CD137)scFv being preferred.

[0119] The DNA and protein sequences of the light chain VL of CD137scFv are sequence numbers 9 and 10, and the DNA and protein sequences of the heavy chain VH of CD137scFv are sequence numbers 11 and 12.

[0120] Therefore, component (b) is preferably encoded by a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, and 100% (in increasing order of priority) identical to sequence numbers 10 and / or 12, or by a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, and 100% (in increasing order of priority) identical to sequence numbers 9 and / or 11.

[0121] The DNA and protein sequences of the light chain VL of NKp46scFv are sequence numbers 51 and 52, and the DNA and protein sequences of the heavy chain VH of NKp46scFv are sequence numbers 53 and 54.

[0122] Therefore, component (b) is preferably encoded by a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, and 100% (in increasing order of priority) identical to sequence numbers 52 and / or 54, or by a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, and 100% (in increasing order of priority) identical to sequence numbers 51 and / or 53.

[0123] The DNA and protein sequences of the light chain VL of NKG2DscFv are sequence numbers 55 and 56, and the DNA and protein sequences of the heavy chain VH of NKG2DscFv are sequence numbers 57 and 58.

[0124] Therefore, component (b) is preferably encoded by a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, and 100% (in increasing order of priority) identical to sequence numbers 56 and / or 58, or by a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, and 100% (in increasing order of priority) identical to sequence numbers 55 and / or 57.

[0125] The DNA and protein sequences of the light chain (VL) of CD137scFvdss are sequence numbers 59 and 60, and the DNA and protein sequences of the heavy chain (VH) of CD137scFvdss are sequence numbers 61 and 62.

[0126] Therefore, component (b) is preferably encoded by a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, and 100% (in increasing order of priority) identical to sequence numbers 60 and / or 62, or by a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, and 100% (in increasing order of priority) identical to sequence numbers 59 and / or 61.

[0127] The analysis and alignment of nucleotide and amino acid sequences related to the present invention are preferably performed using the NCBI BLAST algorithm (Stephen F. Altschul, Thomas L. Madden, Alejandro A. Schaffer, Jinghui Zhang, Zheng Zhang, Webb Miller, and David J. Lipman (1997), Nucleic Acids Res. 25:3389-3402). BLAST can be used for nucleotide sequences (nucleotide BLAST) and amino acid sequences (protein BLAST). Those skilled in the art are aware of additional programs suitable for nucleic acid sequence alignment.

[0128] According to another preferred embodiment of the first aspect of the present invention, components (a), (b), and / or (c) are fused to one another by a mobile linker, preferably a mobile peptide linker, most preferably a mobile peptide linker of at least five amino acids.

[0129] The peptide linker is preferably a short amino acid sequence in the range of 5 to 50 amino acids. The peptide linker is preferably a GS linker. The GS linker consists only of glycine and serine amino acids.

[0130] A preferred example of a GS linker is selected from seven linkers, sequence numbers 13-17, 63, and 64, which are encoded by the nucleic acid sequences sequence numbers 18-22, 65, and 66.

[0131] According to a more preferred embodiment of the first aspect of the present invention, component (a) is located at the N-terminus of the fusion protein, component (b) is located between components (a) and (c), and portion (c) is located at the C-terminus.

[0132] This particular orientation of components (a), (b), and (c) is illustrated by the examples. The orientation of components (a), (b), and (c) is not particularly limited, but in particular, this orientation was used in the examples and found to be sufficiently functional.

[0133] According to a more preferred embodiment of the first aspect of the present invention, the fusion protein further comprises a purification tag, preferably a His tag or a myc tag.

[0134] Purification tags (or affinity tags) are attached to proteins so that they can be purified from their crude biological source using affinity techniques (e.g., affinity chromatography). These include chitin-binding proteins (CBPs), maltose-binding proteins (MBPs), Strep tags, glutathione-S-transferase (GST), polyhistidines (His), and myc.

[0135] His tags or myc tags are preferred because they are used in the examples. Polyhistidine tags are amino acid motifs in proteins that typically consist of at least six histidine (His) residues and are often located at the N-terminus or C-terminus of a protein. Polyhistidine tags are most commonly simply called His tags. Myc tags are polypeptide protein tags derived from the c-myc gene product and can be attached to proteins using recombinant DNA technology.

[0136] The His tag preferably has the amino acid sequence of SEQ ID NO: 23 or is coded by SEQ ID NO: 24. The myc tag preferably has the amino acid sequence of SEQ ID NO: 25 or is coded by SEQ ID NO: 26. Linkers are also conceivable that contain, or are coded by, sequences having at least 80%, preferably at least 90%, and most preferably at least 95% sequence identity with any one of SEQ ID NOs. 23-26.

[0137] According to a more preferred embodiment of the first aspect of the present invention, the antigen / target of (a) 4-1BB, NKG2D, NKp30, NKp46, NKp44, 2B4, CD28 or DNAM-1, (b) IL-15, IL-2, IL-18, IL-21 or IL-12, and / or (c) is a human antigen / target.

[0138] The use of human targets / antigens is particularly advantageous for the proliferation of human NK cells, T cells, and / or NTK cells. The proliferation of human NK cells, T cells, and / or NTK cells is of particular interest because it can be used in NK / T / NTK cell-based therapeutic strategies such as cancer treatment or adoptive immunotherapy (see the overview in Rezani and Rouce (2015), Front.Immunol.).

[0139] According to another preferred embodiment of the first aspect of the present invention, component (c) comprises IL-15 fused to the sushi domain of the IL-15 receptor.

[0140] The Sushi domain of the soluble IL-15 receptor alpha is essential and sufficient for binding to IL-15 (see Xq et al. (2001), J Immunol;167(1):277-82). The Sushi domain is a common motif in protein-protein interactions. The Sushi domain is also known as a short consensus repeat or type 1 glycoprotein motif. These have been identified in several protein-binding molecules, including the complement components C1r, C1s, factor H, and C2m, as well as the non-immune molecules factor XIII and β2 glycoprotein. A typical Sushi domain has approximately 60 amino acid residues and contains four cysteine. The first cysteine ​​forms a disulfide bond with the third cysteine, and the second cysteine ​​forms a disulfide bridge with the fourth cysteine. The two disulfide bonds are essential for maintaining the protein's tertiary structure. In the exemplary fusion protein of the present invention used in the attached examples, the sushi domain of the IL-15 receptor is therefore also preferred to be used to bind IL-15 to the remaining components of the fusion protein.

[0141] The binding of IL-15 to the sushi domain of IL-15R is known to suppress inflammatory and allogeneic responses. This suppression may be advantageous for in vivo applications, for example, to prevent side effects.

[0142] In this specification, IL-15 preferably comprises the amino acids of SEQ ID NO: 27 or is encoded by SEQ ID NO: 28.

[0143] The sushi domain of IL-15R preferably contains the amino acids of SEQ ID NO: 29 or is encoded by SEQ ID NO: 30.

[0144] Sequences having at least 80%, preferably at least 90%, and most preferably at least 95% sequence identity with any one of sequence numbers 27-30 are also conceivable.

[0145] According to another preferred embodiment of the first aspect of the present invention, component (c) comprises IL-2.

[0146] In this specification, IL-2 preferably comprises the amino acids of SEQ ID NO: 67 or is encoded by SEQ ID NO: 68.

[0147] Sequences having at least 80%, preferably at least 90%, and most preferably at least 95% sequence identity with sequence number 67 or 68 are also conceivable.

[0148] According to a more preferred embodiment of the first aspect of the present invention, the fusion protein comprises (a) the amino acid sequence of SEQ ID NOs: 31, 33, 35, 37, 69, 71, 73, 75 or 77, (b) the amino acid sequence encoded by SEQ ID NOs: 32, 34, 36, 38, 70, 72, 74, 76 or 78, and (c) at least 80%, at least 85%, at least 90%, at least 95% of SEQ ID NOs: 31, 33, 35, 37, 69, 71, 73, 75 or 77. (d) an amino acid sequence that is identical to at least 98%, at least 99%, and 100% (in increasing order of priority), or an amino acid sequence encoded by a nucleotide sequence that is identical to at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, and 100% (in increasing order of priority) SEQ ID NOs. 32, 34, 36, 38, 70, 72, 74, 76, or 78, or comprises or consists of such sequences.

[0149] Sequence ID 31 is the amino acid sequence of the exemplary fusion protein RTX-CD137scFv-IL-15, and Sequence ID 32 is its nucleic acid sequence.

[0150] Sequence ID 33 is the amino acid sequence of the exemplary fusion protein BCMA-CD137scFv-IL-15, and Sequence ID 34 is its nucleic acid sequence.

[0151] Sequence ID 35 is the amino acid sequence of the exemplary fusion protein RTX-DuoFab-CD137scFv-IL-15, and Sequence ID 36 is its nucleic acid sequence.

[0152] Sequence ID 37 is the amino acid sequence of the exemplary fusion protein BCMA-DuoFab-CD137scFv-IL-15, and Sequence ID 38 is its nucleic acid sequence.

[0153] Sequence ID 69 is the amino acid sequence of the exemplary fusion protein CD19-CD137scFv-IL-15, and Sequence ID 70 is its nucleic acid sequence.

[0154] Sequence ID 71 is the amino acid sequence of the exemplary fusion protein RTX-CD137scFvdss-IL-15, and Sequence ID 72 is its nucleic acid sequence.

[0155] Sequence ID 73 is the amino acid sequence of the exemplary fusion protein RTX-CD137scFv-IL-2, and Sequence ID 74 is its nucleic acid sequence.

[0156] Sequence ID 75 is the amino acid sequence of the exemplary fusion protein RTX-NKp46scFv-IL-15, and Sequence ID 76 is its nucleic acid sequence.

[0157] Sequence ID 77 is the amino acid sequence of the exemplary fusion protein RTX-NKG2DscFv-IL-15, and Sequence ID 78 is its nucleic acid sequence.

[0158] The exemplary fusion protein described above also includes, in addition to components (a) to (c) and the linker, a cleavage hinge region, a CH1 region, and a spacer sequence.

[0159] The cleavage-type hinge region has the amino acid sequence of SEQ ID NO: 39 and the nucleic acid sequence of SEQ ID NO: 40.

[0160] The CH1 region has the amino acid sequence of SEQ ID NO: 41 and the nucleic acid sequence of SEQ ID NO: 42.

[0161] Spacer sequence 1 has the amino acid sequence of sequence number 43 and the nucleic acid sequence of sequence number 44. Spacer sequence 2 has the amino acid sequence of sequence number 45 and the nucleic acid sequence of sequence number 46.

[0162] In a second aspect, the present invention relates to a nucleic acid sequence, a set of nucleic acid molecules, an expression vector, or a set of expression vectors that encode the fusion protein of the first aspect.

[0163] The term “nucleic acid molecule” in accordance with the present invention includes DNA, e.g., cDNA or double-stranded or single-stranded genomic DNA, and RNA. In this context, “DNA” (deoxyribonucleic acid) means any strand or sequence of chemical components called nucleotide bases, adenine (A), guanine (G), cytosine (C), and thymine (T), linked on a deoxyribose sugar backbone. DNA can have one nucleotide base strand or two complementary strands that can form a double helix structure. RNA is also included. “RNA” (ribonucleic acid) means any strand or sequence of chemical components called nucleotide bases, adenine (A), guanine (G), cytosine (C), and uracil (U), linked on a ribose sugar backbone. RNA usually has one nucleotide base strand, e.g., mRNA. Single-stranded and double-stranded hybrid molecules, i.e., DNA-DNA, DNA-RNA, and RNA-RNA, are also included. Nucleic acid molecules may be modified by many means known in the art. Non-limiting examples of such modifications include methylation, "caps," substitution with one or more naturally occurring nucleotide analogs, and internucleotide modifications, such as those by uncharged bonds (e.g., methylphosphonates, phosphotriesters, phosphoramidates, carbamates, etc.) and charged bonds (e.g., phosphorothioates, phosphorodithioates, etc.). Nucleic acid molecules, also referred to hereafter as polynucleotides, may contain one or more additional covalent bonds, such as proteins (e.g., nucleases, toxins, antibodies, signal peptides, poly-L-lysine, etc.), intercalators (e.g., acridine, psoralens, etc.), chelating agents (e.g., metals, radioactive metals, iron, metal oxides, etc.), and alkylating agents. Polynucleotides can be derivatized by the formation of methyl or ethyl phosphotriester bonds or alkylphosphoamidate bonds. Furthermore, nucleic acid mimetic molecules known in the art, such as synthetic or semi-synthetic derivatives of DNA or RNA, and mixed polymers are also included.Such nucleic acid mimetic molecules or nucleic acid derivatives according to the present invention include phosphorothioate nucleic acids, phosphoramidate nucleic acids, 2'-O-methoxyethyl ribonucleic acid, morpholino nucleic acids, hexitol nucleic acids (HNA), peptide nucleic acids (PNA), and locked nucleic acids (LNA) (see Braasch and Corey, Chem Biol 2001, 8:1). LNA is an RNA derivative in which the ribose ring is bound by a methylene bond between the 2'-oxygen and 4'-carbon. Also included are nucleic acids containing modified bases, such as thiouracil, thioguanine, and fluorouracil. Nucleic acid molecules typically contain genetic information, including information used by cellular mechanisms to create proteins and / or polypeptides. The nucleic acid molecules of the present invention may include promoters, enhancers, response elements, signal sequences, polyadenylated sequences, introns, and 5'- and 3'-noncoding regions.

[0164] As described above, the nucleic acid molecule according to the present invention encodes the fusion protein of the present invention. The fusion protein of the present invention may also be encoded by a set of nucleic acid molecules, preferably a set of two nucleic acid molecules. This is because the antibody or fragment thereof contained in the fusion protein (e.g., full-length antibody, scFv, or Fab) contains heavy and light chain sequences that, when expressed, for example, in a cell, self-assemble to become an antibody. The heavy and light chain sequences can be encoded by a set of different nucleic acid molecules, preferably a set of two nucleic acid molecules.

[0165] The term “vector” in the present invention preferably means a plasmid, cosmid, virus, bacteriophage, or other vector that encodes the fusion protein of the present invention in an expressible form, for example, a plasmid, cosmid, virus, bacteriophage, or other vector conventionally used in genetic engineering. For the same reasons discussed with respect to the set of nucleic acid molecules of the present invention, the fusion protein of the present invention may be encoded by a set of vectors, preferably a set of two vectors.

[0166] The nucleic acid molecule encoding the fusion protein of the present invention can be inserted into, for example, several commercially available vectors. Non-limiting examples include prokaryotic plasmid vectors such as the pUC series, pBluescript (Stratagene), pET series expression vectors (Novagen), or pCRTOPO (Invitrogen), and vectors suitable for expression in mammalian cells, such as pREP (Invitrogen), pSec Tag2 (Invitrogen), pcDNA3 (Invitrogen), pCEP4 (Invitrogen), pMC1neo (Stratagene), pXT1 (Stratagene), pSG5 (Stratagene), EBO-pSV2neo, pBPV-1, pdBPVMMTneo, pRSVgpt, pRSVneo, pSV2-dhfr, pIZD35, pLXIN, pSIR (Clontech), pIRES-EGFP (Clontech), pEAK-10 (Edge Biosystems), pTriEx-Hygro (Novagen), and pCINeo (Promega). Examples of plasmid vectors suitable for Pichia pastoris include plasmids pAO815, pPIC9K, and pPIC3.5K (all from Invitrogen).

[0167] Nucleic acid molecules to be inserted into the vector can be synthesized, for example, by standard methods or isolated from natural sources. Ligation of coding sequences to transcriptional regulatory elements and / or other amino acid coding sequences can also be performed using established methods. Transcriptional regulatory elements (part of the expression cassette) that ensure expression in prokaryotes or eukaryotic cells are well known to those skilled in the art. These elements include regulatory sequences that ensure the initiation of transcription (e.g., translation start codons, promoters, e.g., naturally related or heterologous promoters and / or insulators; see above), internal ribosome entry sites (IRES) (Owens, Proc. Natl. Acad. Sci. USA 98(2001), 1471-1476), and optionally, poly-A signals that ensure the termination of transcription and stabilization of the transcript. Additional regulatory elements may include transcriptional enhancers and translational enhancers. Preferably, the polynucleotide encoding the fusion protein of the present invention is operably ligated to such expression regulatory sequences to enable expression in prokaryotes or eukaryotic cells. The vector may further include a nucleic acid sequence encoding a secretion signal as a further regulatory element. Such sequences are well known to those skilled in the art. Furthermore, depending on the expression system used, a leader sequence capable of directing the expressed polypeptide into an intracellular compartment may be added to the polynucleotide coding sequence of the present invention. Such leader sequences are well known in the art.

[0168] Furthermore, the vector preferably contains a selectable marker. Examples of selectable markers include genes encoding resistance to neomycin, ampicillin, hygromycin, and kanamycin. Specially designed vectors enable DNA shuttering between different hosts, such as between bacterial and fungal cells, or between bacterial and animal cells (e.g., Gateway systems available from Invitrogen). Expression vectors according to the present invention can direct the replication and expression of the polynucleotides and encoded peptides or fusion proteins of the present invention. In addition to introduction via vectors such as phage vectors or viral vectors (e.g., adenoviruses, retroviruses), the nucleic acid molecules described herein above may be designed for direct introduction into cells or introduction via liposomes. Furthermore, baculovirus systems, or systems based on vaccinia virus or Semryki forest virus, can be used as eukaryotic expression systems for the nucleic acid molecules of the present invention.

[0169] In a third aspect, the present invention relates to a host cell, preferably a non-human host cell, containing the nucleic acid molecule or expression vector of the second aspect.

[0170] The term "host cell" means any cell of any organism that is selected, modified, transformed, grown, or otherwise used or manipulated for the cellular production of the fusion protein of the present invention. Therefore, host cells are generally ex vivo or in vitro cells and / or isolated cells.

[0171] The host cells of the present invention are typically generated by introducing the nucleic acid molecule or vector of the present invention into the host cell, and in its presence mediates the expression of the nucleic acid molecule of the present invention that encodes the fusion protein of the present invention. The host from which the host cells are derived or isolated may be any prokaryotic or eukaryotic cell or organism, except preferably human embryonic stem cells obtained directly by disruption of a human embryo.

[0172] Suitable prokaryotes (bacteria) useful as hosts for the present invention include, for example, Escherichia coli (E. coli) (e.g., E. coli strains BL21, HB101, DH5a, XL1 Blue, Y1090 and JM101), Salmonella typhimurium, Serratia marcescens, Burkholderia glumae, Pseudomonas putida, Pseudomonas fluorescens, Pseudomonas stutzeri, Streptomyces lividans, Lactococcus lactis, and Mycobacterium smegmatis. These are commonly used for cloning and / or expression, such as *Streptomyces smegmatis*, *Streptomyces coelicolor*, or *Bacillus subtilis*. Appropriate culture media and conditions for the above host cells are well known in the art.

[0173] Suitable eukaryotic host cells may be vertebrate cells, insect cells, fungal / yeast cells, nematode cells, or plant cells. Fungal / yeast cells may be Saccharomyces cerevisiae cells, Pichia pastoris cells, or Aspergillus cells. Preferred examples of host cells to be genetically engineered using the nucleic acid molecules or vectors of the present invention are cells of yeast, Escherichia coli (E. coli), and / or Bacillus species (e.g., B. subtilis). In one preferred embodiment, the host cell is a yeast cell (e.g., S. cerevisiae).

[0174] In another preferred embodiment, the host cells are mammalian host cells such as Chinese hamster ovary (CHO) cells, mouse myeloma lymphoblastoid cells, human fetal kidney cells (HEK-293), human fetal retinal cells (Crucell's Per.C6), or human amniotic cells (Glycotope and CEVEC). These cells are frequently used in the art to produce recombinant proteins. CHO cells are the most commonly used mammalian host cells for the industrial production of human recombinant protein therapies.

[0175] The present invention also relates to transgenic animals, preferably non-human transgenic animals, that contain the vector of the present invention.

[0176] Transgenic animals can be used for antibody production, as outlined, for example, in Brueggemann (2015), Arch Immunol Ther Exp(Warsz); 63(2):101-108. The transgenic animals are preferably non-human mammals. The antibodies may be produced so that they can be obtained from the milk of the transgenic mammal. Therefore, the mammals are preferably goats, sheep, or cows.

[0177] In a fourth aspect of the present invention, the present invention relates to a method for producing a fusion protein of the first aspect, comprising (a) culturing a host cell of the third aspect under conditions in which the host cell expresses a fusion protein of the first aspect, and (b) isolating the fusion protein of the first aspect expressed in (a).

[0178] The term "culture" refers to the process of growing host cells under controlled conditions. These conditions can vary depending on the host cells used. Those skilled in the art are well aware of the methods for establishing optimized culture conditions. Furthermore, methods for establishing, maintaining, and manipulating cell cultures are extensively described in state-of-the-art technologies.

[0179] Methods for isolating the fusion protein of the present invention are well known in the art and include, but are not limited to, method steps such as ion exchange chromatography, gel filtration chromatography (size exclusion chromatography), affinity chromatography, high-performance liquid chromatography (HPLC), reverse-phase HPLC, disk gel electrophoresis, or immunoprecipitation. For example, see Antibody Purification Handbook, GE Healthcare, 18-1037-46.

[0180] The fusion protein of the present invention expressed in (a) according to the present invention refers to the product of the process, meaning that information from the nucleic acid molecule encoding the fusion protein of the present invention in the host cell can induce a process used in the synthesis of the fusion protein of the present invention. Several steps in this process, such as transcription, RNA splicing, translation, and post-translational modification of the fusion protein of the present invention, can be regulated by methods known in the art. Thus, such regulation can enable control over the timing, location, and amount of the fusion protein produced.

[0181] In a fifth aspect, the present invention relates to a composition, preferably a pharmaceutical composition or kit, comprising a fusion protein, nucleic acid molecule, set of nucleic acid molecules, expression vector, set of expression vectors, or host cell according to the above aspects of the present invention.

[0182] The composition of the present invention comprises a fusion protein, nucleic acid molecule, set of nucleic acid molecules, expression vector or set of expression vectors, or host cell according to the above-described embodiment of the present invention, and preferably at least one further component, such as a solvent, carrier, or excipient.

[0183] According to the present invention, the term “pharmaceutical composition” refers to a composition for administration to a patient, preferably a human patient. The pharmaceutical compositions of the present invention include a fusion protein, nucleic acid molecule, set of nucleic acid molecules, expression vector or set of expression vectors, or host cells according to the above embodiments of the present invention. Optionally, further molecules may be included that can alter the properties of the compound of the present invention, thereby stabilizing, regulating, and / or activating its function, for example. The composition may be in solid, liquid, or gaseous form, and may be, in particular, in the form of a powder, tablet, solution, or aerosol. The pharmaceutical compositions of the present invention may optionally and additionally include a pharmaceutically acceptable carrier. Examples of suitable pharmaceutical carriers are well known in the art and include phosphate-buffered saline, water, emulsions such as oil / water emulsions, various types of wetting agents, sterile solutions, organic solvents including DMSO, and the like. Compositions containing such carriers can be formulated by well-known conventional methods. These pharmaceutical compositions can be administered to a subject in an appropriate dose. The administration plan will be determined by the attending physician and clinical factors. As is well known in the medical field, the dosage for any given patient depends on many factors, including the patient's size, body surface area, age, the specific compound being administered, sex, time and route of administration, overall health status, and other drugs administered concurrently. The therapeutically effective dose for a given condition is readily determined by routine experiments and is within the scope of the usual skills and judgment of a clinician or physician. Generally, the dosage regimen for this pharmaceutical composition as a regular administration should be in the range of 1 μg to 5 g units / day. However, a more preferable dosage may be in the range of 0.0001 mg to 100 mg / kg body weight per day, even more preferably 0.01 mg to 50 mg / kg body weight, and most preferably 20 mg to 50 mg / kg body weight. The treatment period required to observe changes and the interval until a response occurs after treatment will vary depending on the desired effect. The specific amount can be determined by conventional tests well known to those skilled in the art.

[0184] The compositions of the present invention include a fusion protein, nucleic acid molecule, set of nucleic acid molecules, expression vector or set of expression vectors, or host cells according to the above embodiments of the present invention, and preferably a method of using the kit; i.e., instructions for a method of using the fusion protein to proliferate NK cells, T cells and / or NTK cells.

[0185] In a sixth aspect, the present invention relates to a fusion protein, nucleic acid molecule, set of nucleic acid molecules, expression vector or set of expression vectors, or host cell according to the above aspects of the present invention, optionally combined with CAR NK cells or CAR T cells for use in the treatment of tumors.

[0186] When a fusion protein, nucleic acid molecule, set of nucleic acid molecules, expression vector or set of expression vectors, or host cells according to the above embodiments of the present invention are administered to a target, NK cells, T cells and / or NTK cells proliferate in the target. In this embodiment, component (a) is preferably an antibody or antibody fragment that binds to an antigen expressed on the surface of target cells of tumor cells, such as NK cells, T cells and / or NTK cells. More preferably, the antigen can be found on the surface of tumor cells in the target being treated. The target is a mammal, primate and human, with increasing priority in that order.

[0187] Autologous CAR (chimeric antigen receptor)-NK cell therapy or CAR T cell therapy involves several steps. First, NK cells or T cells are isolated from the patient's or donor's blood. Next, CAR-coding genes are introduced into the NK cells or T cells using (primarily) viral vectors. The CAR-NK cells or T cells are proliferated until a sufficient number of cells are obtained and then adopted into the patient to fight malignant cells. Before injecting the CAR-NK cells or T cells, lymphodepletion is commonly performed in most therapeutic settings to enable efficient cell transplantation. It is important to mention that CAR-NK cells or T cells offer the potential to be an "off-the-shelf" product.

[0188] In a seventh aspect of the present invention, a method for growing NK cells, T cells and / or NTK cells ex vivo or in vitro, comprising the steps of (a) co-culturing NK cells, T cells and / or NTK cells with target cells of NK cells, T cells and / or NTK cells, preferably B cells or tumor cells, in the presence of a fusion protein, nucleic acid sequence, set of nucleic acid sequences, expression vector, set of expression vectors, or host cells according to the above aspects of the present invention, and (b) optionally purifying or isolating the grown NK cells, T cells and / or NTK cells obtained in step (a) from the co-culture.

[0189] According to a preferred embodiment of the seventh aspect, the NK cells, T cells and / or NTK cells are purified NK cells, T cells and / or NTK cells, which are contained in PBMCs, derived from iPSCs, or are CAR NK cells or T cells.

[0190] As described above, in CAR-NK cell or T cell therapy, NK cells or T cells, such as purified NK cells or T cells, NK cells or T cells contained in PBMCs, or CAR NK cells or T cells, need to be proliferated ex vivo or in vitro before being administered to the subject to be treated, such as a subject with a tumor. The method of the seventh embodiment is particularly well suited for the proliferation of such NK cells, T cells and / or NTK cells. As described above, the attached examples show that proliferation rates between 10 and 10,000 times were achieved with the fusion protein of the present invention.

[0191] According to a more preferred embodiment of the seventh aspect, NK cells, T cells and / or NTKs may be pre-cultured (e.g., for 12-24 hours) with IL-12, IL-15 and / or IL-18 prior to step (a). This pre-culture step polarizes the cells into a memory-like phenotype.

[0192] With respect to embodiments characterized in this specification, and in particular in the claims, each embodiment referred to in a dependent claim is intended to be combined with each embodiment of the claim (independent or dependent) to which the dependent claim depends. For example, in the case of independent claim 1 describing three options A, B and C, dependent claim 2 describing three options D, E and F, and claim 3 dependent on claims 1 and 2 and describing three options G, H and I, it should be understood that, unless otherwise specifically mentioned, this specification expressly discloses embodiments corresponding to combinations of A, D, G; A, D, H; A, D, I; A, E, G; A, E, H; A, E, I; A, F, G; A, F, H; A, F, I; B, D, G; B, D, H; B, D, I; B, E, G; B, E, H; B, E, I; B, F, G; B, F, H; B, F, I; C, D, G; C, D, H; C, D, I; C, E, G; C, E, H; C, E, I; C, F, G; C, F, H; C, F, I.

[0193] Similarly, even if an independent claim and / or dependent claim does not specify alternatives, it is understood that if a dependent claim refers to multiple prior claims, all combinations of subject matter covered by them are clearly disclosed. For example, in the case of independent claim 1, dependent claim 2 referring to claim 1, and dependent claim 3 referring to both claim 2 and claim 1, the subject matter combination of claim 3 and 1 is as obvious and clearly disclosed as the subject matter combination of claim 3, 2 and 1. If there is a further dependent claim 4 referring to any one of claims 1-3, the subject matter combinations of claim 4 and 1, claim 4, 2 and 1, claim 4, 3 and 1, and claim 4, 3, 2 and 1 are also obvious and clearly disclosed.

[0194] The above considerations apply to all of the attached claims, with necessary modifications.

[0195] The drawings are as follows: [Brief explanation of the drawing]

[0196] [Figure 1]Design, conception, purification, and biochemical characterization of the fusion protein. (A) Structural scheme of the RTX-CD137scFv-IL-15 fusion; cDNA sequences encoding CD20-VH, CD20-VL, immunoglobulin heavy chain and light chain constant regions from the CD20 antibody rituximab; cDNA sequences encoding variable heavy chain and light chain constant regions to construct scFv with specificity for CD137VL, CD137VH, and CD137(4-1BB); cDNA sequences encoding the sushi domain, hIL-15, sushi domain and human interleukin 15. (B) Mechanism of action. (C) Evaluation of the purity and integrity of the fusion protein by SDS-PAGE and Coomassi staining. Reduced and unreduced protein samples. (D-E) Specific antigen binding of the fusion protein. Dose-dependent binding of fusion proteins (RTX-CD137scFv-IL-15 - red filled circle; RTX-CD137scFv - blue filled square; RTX-IL-15 - light purple filled diamond; 4D5-CD137scFv-IL-15 - black circle; IL-15 - black triangle; trastuzumab - black filled triangle) to CD20-positive Ramos cells (D) and 4-1BB (CD137)-positive CRRF-CEM cells (E). The mean fluorescence value at saturation concentration for each cell line was set to 100%, and all other experimental values ​​were normalized to this value. Data are presented as the mean of three independent measurements, and error bars represent ±SEM. *=<0.05. (F) To determine the functionality of IL-15 in various antibody derivatives (RTX-CD137scFv-IL-15 - red filled circle; RTX-CD137scFv-blue filled square; RTX-IL-15-light purple filled square; 4D5-CD137scFv-IL-15-black circle; IL-15-black filled triangle), serial dilutions of the fusion protein were incubated with CTLL-2 cells. After 48 hours, MTT reagent was added. The mean fluorescence value at the saturation concentration of IL-15 was set to 100%, and all other experimental values ​​were normalized to this value. Data are presented as the mean of four independent measurements, and error bars represent ±SEM; * = < 0.05. [Figure 2]Proliferative capacity of the fusion protein. (A) Proliferation rate of 19 independent NK cell proliferations. (B) To compare the proliferative capacity of the fusion protein containing all structural components and a commercially available proliferation kit, newly isolated NK cells from healthy donors were co-incubated with RTX-CD137scFv-IL-15 and B cells. RTX-CD137scFv-IL-15+B cells - red filled circle. Proliferation beads + IL-2 (beads + IL-2 - gray diamond). (C) To determine the effect of different structural components and the presence of target cells, newly isolated NK cells from healthy donors were co-incubated with the fusion protein and B cells (RTX-CD137-IL-15+B cells - red filled circle; RTX-CD137scFv - blue filled square; RTX-IL-15 - light purple filled square; RTX-CD137scFv-IL-15-15 - black / gray circle). Cells were cultured for up to 4 weeks. Every 3-4 days, the number of NK cells was determined, and fresh medium containing the respective fusion protein was added. The x-fold proliferation of NK cells was plotted against time (days). Data are presented as the mean of three independent measurements, and error bars represent ±SEM. *=<0.05. [Figure 3] Characterization of in vitro proliferating NK cells from healthy donors. (A) The Fc receptor FcγRIIIa (CD16a) plays a crucial role in antibody-dependent cell-mediated cytotoxicity. NK cells (CD56+, CD3-) express different amounts of the Fc receptor FcγRIIIa (CD16a) depending on their activation state. To evaluate this expression, flow cytometry analysis was performed on days 0 and 7 using commercially available CD16, CD56, and CD3 antibodies. The data show representative images of three independent measurements. (B) To characterize the state of NK cells, the expression of specific NK cell markers (CD44; NKp46; NKp44; NKp30; CD69; NKG2D; DNAM-1; CD11a) was determined. Expression was determined by flow cytometry analysis at the start and end of the proliferation period. Data are presented as the mean of three independent measurements, and error bars represent ±SEM. *=<0.05. [Figure 4]Innate cytotoxicity of proliferating NK cells against tumor cells and non-malignant B cells. The innate cytotoxicity of NK cells proliferated by recombinant fusion proteins was determined by measuring NK cell-mediated tumor cell lysis. The 4-hour 51Cr release assay was performed without antibody addition, using proliferating NK cells as effector cells and various tumor cell lines as target cells. (A) NK cell-dependent lysis of K562 cells. Proliferating NK cells were used as effector cells in various effector-to-target cell (E:T) ratios. Data represent the mean + / - SEM of three NK cell donors. (B) 51Cr release assay was performed using tumor cells representing different tumor entities or non-malignant B cells. NK cells were used in a fixed E:T ratio of 10:1. The presented data represent the mean + / - SEM of three NK cell donors. [Figure 5]Antibody-dependent cell-mediated cytotoxicity of proliferating NK cells against tumor cells and non-malignant B cells. (A) Comparison of innate cytotoxicity and antibody-dependent cell-mediated cytotoxicity of NK cells proliferated by recombinant fusion protein. Lysis of tumor cells was determined by performing a 4-hour 51Cr release assay with or without the addition of 5 μg / ml antibody, using proliferating NK cells as effector cells and various cell lines of B-cell malignancies (Granta-519, Raji, SEM, Carnaval, SUDHL-4) or non-malignant B cells (autologous) as target cells. The E:T ratio was 10:1. (B) The effect of antibody-mediated tumor cell lysis was determined by performing a 4-hour 51Cr release assay with or without the addition of 5 μg / ml antibody, using proliferating NK cells as effector cells and various cell lines of B-cell malignancies (Granta-519, Raji, SEM, Carnaval, SUDHL-4) as target cells. The E:T ratio was 10:1. (C) Cytolysis of CD20+ tumor cells (allogeneic) or autologous non-malignant B cells as effector cells using NK cells grown with recombinant fusion protein as effector cells at different effector-to-target (E:T) cell ratios. ADCC was measured in a standard 4-hour chromium release assay with each antibody added at 1 μg / ml. (D) Comparison of ADCC efficacy of NK cells grown differently. NK cells were grown with either microbeads + IL-2 or recombinant fusion protein (RTX-CD137scFv-IL-15) and used as effector cells at different E:T ratios in a 4-hour 51Cr release assay with each antibody at 1 μg / ml. Data represent the mean + / - SEM of three NK cell donors. [Figure 6]Proliferation of NK cells from multiple myeloma patients. (A) To determine the proliferative capacity of various fusion proteins, mononuclear cells from BM aspirate / peripheral blood of multiple myeloma (MM) patients were co-incubated with fusion proteins containing all structural components or fusion proteins lacking one component. Cells were cultured for up to 4 weeks. Every 3-4 days, the cell count was determined and fresh medium containing each fusion protein was added. (B) Characterization of in vitro proliferating NK cells from BM aspirate / peripheral blood of multiple myeloma (MM) patients. To evaluate the expression status of NK cells proliferated with recombinant fusion proteins from BM aspirate / peripheral blood of multiple myeloma (MM) patients, flow cytometry analysis was performed on days 0 and 16 using commercially available CD16, CD56, and CD3 antibodies. A histogram of one exemplary flow cytometry analysis and quantitative values ​​± SEM from three independent experiments are shown. (C) ADCC experiments using multiple myeloma cell lines (n=3, allogeneic) or primary tumor cells (exemplary, autologous) from BM aspirate / peripheral blood of multiple myeloma (MM) patients as target cells. NK cells proliferated with recombinant fusion protein from BM aspirate / peripheral blood of multiple myeloma (MM) patients were used as effector cells, with different effector-to-target (E:T) cell ratios (allogeneic) or an E:T ratio of 10:1 (autologous). ADCC was measured in a standard 4-hour chromium-releasing assay with each antibody added at 5 μg / ml. [Figure 7] Design and size exclusion chromatography analysis of monovalent and bivalent antibody derivatives. (A) Schematic diagram of monovalent and bivalent fusion proteins targeting CD20 or BCMA. (B) Size exclusion chromatography of purified proteins was performed using an AEKTA pure25 liquid chromatography system. Relative absorbance of proteins (milliabsorbance units, mAU) was plotted against elution volume (ml). Eluted monomers are marked with boxes. [Figure 8]Analysis of molecular weight and purity of bivalent antibody derivatives by SDS-PAGE, Coomassie blue staining, and Western blotting. The purity and molecular weight of isolated monomers and polymers were determined by SDS-PAGE and Coomassie blue staining or Western blotting. The protein fraction collected before size exclusion chromatography served as a control (BP). 3 μg of eluted protein fraction, isolated monomers, and polymers were subjected to SDS-PAGE analysis. In Western blotting analysis, heavy chain derivatives were detected using polyhistidine tag-specific antibodies. For light chains, antibodies against the copper constant region were used. BP: Before purification, HC: Heavy chain derivative, LC: Light chain, Mono: Monomer, Multi: Polymer, kDa: Kilodalton. [Figure 9] CD20 binding and surface retention. Flow cytometry analysis was performed using Granta-519 cells to determine the binding ability of RTX-DuoFab-CD137scFv-IL-15 and RTX-CD137scFv-IL-15. (A) CD20 expression in Granta-519 was confirmed. Expression levels were determined using a CD20 antibody. (B+C) Dose-dependent binding of RTX antibody derivatives was analyzed by flow cytometry, and EC50 values ​​were calculated. The highest determined relative mean fluorescence intensity (t0 value of cell surface retention) was set to 100%, and all other values ​​were normalized to this point. The fitted determined relative mean fluorescence intensity (rel.MFI, %) was plotted against protein concentration (nM). (D) In ​​the cell surface retention assay, dissociated molecules in the supernatant were removed at various time points. Molecules remaining on the cell surface were determined by flow cytometry. N=3. Values ​​represent mean + / - SEM. [Figure 10]BCMA binding and surface retention. Flow cytometry analysis was performed using transfected and untransfected Lenti-X cells to determine the binding ability of BCMA-DuoFab-CD137scFv-IL-15 and BCMA-CD137scFv-IL-15 molecules. (A+B) BCMA expression was analyzed in transfected and untransfected cells. (C) Flow cytometry analysis was performed to determine the binding activity of BCMA antibody derivatives. The highest determined relative mean fluorescence intensity (t0 value of cell surface retention) was set to 100%, and all other values ​​were normalized to this point. The fitted determined relative mean fluorescence intensity (rel.MFI, %) was plotted against protein concentration (nM). The results are shown as a hyperbola. n=3. Values ​​represent mean + / - SEM. (D) In ​​the cell surface retention assay, dissociated molecules in the supernatant were removed at various time points. Molecules remaining on the cell surface were determined by flow cytometry. n=1. [Figure 11] CD137 binding analysis. Flow cytometry analysis was performed using stimulated (PMA and ionomycin) and unstimulated CEM cells to determine the CD137 binding ability of antibody derivatives. (A+B) CD137 expression in stimulated and unstimulated cells was analyzed before performing binding analysis. Isotype control (white), CD137 antibody (gray). (C) Dose-dependent binding of antibody derivatives was analyzed. Normalized relative mean fluorescence intensity (MFI, %) was plotted against protein concentration (nM). Results are shown as dose-response curves. n=3. Values ​​represent mean + / - SEM. [Figure 12] Functionality of the IL-15 component of antibody derivatives. To determine the functionality of IL-15 in various antibody derivatives, serial dilutions were prepared and incubated with the IL-15-responsive cell line CTLL2. After 48 hours, MTT reagent was added. After 24 hours, the absorbance of the purple formazan solution was measured. Relative metabolic activity (%) was plotted against concentration (pM). n=3. Values ​​represent mean + / - SEM. [Figure 13]Comparison of the ability of bivalent and monovalent molecules to induce NK cell proliferation. To determine the (target) cell-dependent co-activation of bivalent versus monovalent antibody derivatives, newly isolated NK cells were co-incubated with either B cells (CD20+) and RTX antibody derivatives or INA-6 cells (BCMA+) and BCMA antibody derivatives at various concentrations. Co-culture was performed in 24-well plates for 26 days. Every 3-4 days, the number of NK cells was determined, and fresh medium and antibody derivatives were added. The x-fold proliferation of NK cells was plotted against time (days). n=3. [Figure 14] Cell surface marker expression in NK cells proliferated with antibody derivatives. Various surface markers associated with the activation phenotype were selected. Expression was determined at the start and end of the proliferation period. n=3. [Figure 15] NK cells proliferated with RTX-DuoFab-CD137scFv-IL-15 are not cytotoxic to healthy B cells but mediate ADCC. To determine whether proliferating NK cells can still distinguish between healthy and malignant cells, we analyzed NK cell-mediated target cell lysis by performing a Cr51 release assay using autologous B cells as target cells. n=3. [Figure 16] Cytotoxic activity of proliferating NK cells - Allogeneic ADCC setup using RTX antibody derivatives. To determine the cytotoxic activity of proliferating NK cells, tumor cell lysis mediated by NK cells was measured by performing a Cr51 release assay. Innate cytotoxicity was analyzed, and ADCC reactions were performed using proliferating NK cells as effector cells and Granta519 cells as target cells. Two different concentrations of the monoclonal antibody rituximab (RTX 1 μg / ml and RTX 0.01 μg / ml) were used. Reactions without antibody (BR+NK = innate cytotoxicity) or reactions containing an unrelated antibody control trastuzumab (HER2) served as controls. n=3. [Figure 17]Cytotoxic activity of proliferating NK cells – allogeneic setting using BCMA antibody derivatives. To determine the cytotoxic activity of proliferating NK cells, tumor cell lysis mediated by NK cells was measured by performing a Cr51 release assay. Innate cytotoxicity was analyzed, and ADCC reactions were performed using proliferating NK cells as effector cells and Granta519 cells as target cells. Two different concentrations of the monoclonal antibody rituximab (RTX 1 μg / ml and RTX 0.01 μg / ml) were used. Reactions without antibody (BR+NK=innate cytotoxicity) or reactions containing the unrelated antibody trastuzumab (HER2) served as controls. n=3. [Figure 18] The effect of B cell stimulation frequency on NK cell proliferation. Ex vivo quantification of NK cell proliferation to determine (tumor) cell-dependent co-activation of antibody derivatives. Newly isolated NK cells were co-incubated with the proliferation molecule RTX-CD137scFv-IL-15 or RTX-DuoFab-CD137scFv-IL-15 and B cells (CD20+). Co-culture was performed for 28 days. Every 3-4 days, the number of NK cells was determined, and fresh medium and antibody were added. B cells were added at various time points (once at the start, twice (days 0 and 14), once a week, or twice a week). The x-fold proliferation of NK cells was plotted against time (days). n=3. [Figure 19] The effect of the B cell to NK cell ratio on NK cell proliferation. Quantification of NK cell proliferation ex vivo to determine the (target) cell-dependent co-activation of RTX-CD137scFv-IL-15 or RTX-DuoFab-CD137scFv-IL-15. Newly isolated NK cells were co-incubated with the proliferation molecule RTX-CD137scFv-IL-15 or RTX-DuoFab-CD137scFv-IL-15 and B cells (CD20+). Co-culture was performed for 28 days. Every 3-4 days, the number of NK cells was determined and fresh medium and antibody were added. Different B:NK cell ratios were analyzed. x-fold proliferation of NK cells was plotted against time (days). Each panel represents a different NK cell donor. [Figure 20]T cell proliferation. To analyze whether the novel fusion protein of the present invention can also promote T cell proliferation, MNCs or CD3-positive T cells were isolated from healthy donors by MACS sorting. To determine the effects of different structural components and the presence of target cells, (A) newly isolated MNCs, or (B) a mixture of purified B cells and T cells from healthy donors, were co-incubated with the fusion protein (RTX-CD137scFv-IL-15;RTX-CD137scFv;RTX-IL-15;Her2-CD137scFv-IL-15). Cells were cultured for up to 4 weeks. Fresh medium containing each fusion protein was added every 3-4 days. Growth rates were plotted against time (days). Data are presented as the average of 3 wells from one donor. Proliferated cells were analyzed for T cell content by flow cytometry. [Figure 21] RTX-CD137scFv-IL-15 induces proliferation of γδ T cells. B cells and T cells were isolated from PBMCs of healthy donors and co-cultured in the presence of RTX-CD137scFv-IL-15. The content of vδ1 and vδ2 γδ T cells was analyzed and quantified by multicolor flow cytometry on days 0, 14, and 21. In summary, these data demonstrate that RTX-CD137scFv-IL-15 was able to induce significant proliferation of γδ T cells. A) Data from Donor 1, B) Data from Donor 2. [Figure 22]Proliferation of IL-12, IL-15, and IL-18-stimulated NK cells to impart memory-like properties. When NK cells were cultured overnight with IL-12, IL-15, and IL-18, and then proliferated with RTX-CD137scFv-IL-15 in the presence of B cells (B), and when NK cells were cultured overnight with IL-12, IL-18, and the proliferation molecule RTX-CD137scFv-IL-15, followed by a proliferative phase induced by RTX-CD137scFv-IL-15 in the presence of B cells (C), the proportion of CD56 / CD16 double-positive NK cells was higher compared to NK cells proliferated only with RTX-CD137scFv-IL-15 as a monotherapy agent (A). Note: The proliferative phase was performed in the presence of B cells as described above. To evaluate FcγRIIIa (CD16a) expression, flow cytometry analysis was performed on days 0, 14, and 21 using commercially available CD16, CD56, and antibodies. The data shown represent the results. [Figure 23] A scheme of alternative molecular designs, including the closest competing molecular design. Six additional molecular designs were developed to analyze the effects of the closest competing molecules based on different co-stimulatory receptors, alternative B cell targets, alternative cytokines, disulfide stabilization, and 4-1BB ligands to stimulate CD137 (Kerner, Mol Cancer Ther, 2014; Beha, Mol Cancer Ther, 2019). [Figure 24] The purified novel molecular variants exhibit the expected molecular structure. Novel molecular variants derived from RTX-CD137scFv-IL-15 were generated by transient transfection in CHO-S cells and purified by affinity chromatography and size exclusion chromatography. In SDS-PAGE under reducing or non-reducing conditions, the purified molecules exhibit the expected molecular mass and aggregate structure. [Figure 25]Proliferative capacity of RTX-CD137scFv-IL-15 compared to IL15-RTXscFv-41BB-ligand. A) To compare the proliferative capacity of both molecules, newly isolated NK cells from healthy donors were co-incubated with B cells and each molecule according to the proliferation protocol described above. Comparison of RTX-CD137scFv-IL-15 (red circles) with the competing molecule IL15-RTXscFv-41BB-ligand revealed that, unexpectedly, RTX-CD137scFv-IL-15 exhibited significantly higher proliferative capacity than IL15-RTX-scFv-41BB-ligand, despite targeting the same surface structure on B cells and inducing IL15 receptors and CD137 on NK cells. The x-fold proliferation of NK cells was plotted against time (days). Data are presented as mean values ​​from three independent NK cell donors, and error bars represent ±SEM; *=p<0.05. B) NK cells were labeled with CFSE and left untreated (NK cells only), co-incubated with B cells (NK cells + B cells), or co-incubated with B cells in the presence of RTX-CD137scFv-IL-15 (NK cells + B cells + RTX-CD137scFv-IL-15). After each cell division, the CFSE signal was reduced / diluted to allow for accurate measurement of cell division. After 5 days, CFSE was measured by flow cytometry and cell division was calculated. Cells that had undergone at least one cell division were considered proliferative. Data are presented as mean values ​​from three independent NK cell donors, and error bars represent ±SEM. [Figure 26] Proliferative capacity of proliferation molecule variants containing IL-2 or Il-15. To compare the proliferative capacity of the proliferation molecules, newly isolated NK cells from healthy donors were co-incubated with B cells and each molecule. The culture medium was changed and proteins were supplemented as described above. When IL-2 (RTX-CD137scFv-IL-2, triangle) was used instead of Il-15 (RTX-CD137scFv-IL-15, black circle) in the design of the proliferation molecule of the present invention, a similar ability to induce NK cell proliferation was obtained. x-fold proliferation of NK cells was plotted against time (days). Data are presented as the mean value from three independent NK cell donors, and error bars represent ±SEM. [Figure 27] Proliferative capacity of CD19 or CD20-inducing proliferation molecule variants. To compare the proliferative capacity of proliferation molecules, newly isolated NK cells from healthy donors were co-incubated with B cells and each molecule. Targeting CD19 (CD19-CD137scFv-IL-15, white circles) instead of CD20 (RTX-CD137scFv-IL-15, black circles) in B cells resulted in a decrease in the ability of NK cells to induce proliferation. x-fold proliferation of NK cells was plotted against time (days). Data are presented as mean values ​​from three independent NK cell donors, and error bars represent ±SEM. *=p<0.05. [Figure 28] Proliferative capacity of growth molecules with alternative "co-stimulatory" activity. To compare the proliferative capacity of growth molecules, newly isolated NK cells from healthy donors were co-incubated with B cells and each molecule. Stimulation via CD137 (RTX-CD137scFv-IL-15, circle, ●), NKG2D (RTX-NKG2DscFv-IL-15, ▽), and NKp46 (RTX-NKp46scFv-IL-15, △). x-fold proliferation of NK cells was plotted against time (days). Data are presented as mean values ​​from three independent NK cell donors, and error bars represent ±SEM. [Figure 29] Proliferative capacity of RTX-CD137scFv-IL15 compared to RTX-CD137scFvdss-IL15. To compare the proliferative capacity of both molecules, newly isolated NK cells from healthy donors were co-incubated with B cells and each molecule. No significant difference was observed when comparing RTX-CD137scFv-IL-15 (circles) and RTX-CD137scFvdss-IL-15 (squares). Therefore, the proliferation capacity of the molecule was not affected even with the inclusion of additional disulfide crosslinks. However, a decrease in multimerization was observed in size exclusion chromatography. The x-fold proliferation of NK cells was plotted against time (days). Data are presented as the mean value from three independent NK cell donors, and error bars represent ±SEM. [Figure 30] Proliferative capacity of proliferation molecules - Overview. All molecules in the novel “Targeted Fab-scFv-Cytokine” designs described herein exhibit superior proliferation rates compared to the closest competing molecule designs in the “Cytokine-Targeted scFv-Natural Ligand” designs. Data are presented as mean values ​​from three independent NK cell donors, and error bars represent ±SEM. [Figure 31] NK cells proliferated with a novel "alternative growth molecule" exhibit potent intrinsic cytotoxicity against sensitive K562 cells. The intrinsic cytotoxicity of NK cells proliferated with the novel fusion protein was analyzed using a classic 4-hour Cr51 assay. K562 cells functioned as the target cells. E:T ratio = 10:1. Data are presented as mean values ​​from three independent NK cell donors, and error bars represent ±SEM. [Figure 32] NK cells proliferated with a novel "proliferation agent molecule" exhibit increased innate cytotoxicity and improved ADCC activity. Innate cytotoxicity and ADCC activity of NK cells proliferated with the novel fusion protein were analyzed using a classic 4-hour Cr51 assay. GRANTA-519 cells, a less sensitive NK cell target, functioned as target cells. E:T ratio = 10:1. Rituximab (with antibody) was added to evaluate the ADCC capacity of the proliferated NK cells (final concentration 1 μg / ml). Data are presented as mean values ​​from three NK cell donors, and error bars represent ±SEM. [Figure 33] NK cells proliferated with a novel "proliferation agent molecule" show increased activation compared to those proliferated with IL15-RTXscFv-41BB-ligand. The expression of typical activation markers CD69, the innate cytotoxic receptor NKp30, and FcγRIIIa (CD16a) was analyzed by flow cytometry in NK cells proliferated with the novel fusion protein or competing molecule. The mean values ​​from three NK cell donors are shown in + / - SEM. [Figure 34]NK cells proliferated with RTX-CD137scFv-IL15 show a higher proportion of FcγRIIIa-highly expressing NK cells. NK cells proliferated with RTX-CD137scFv-IL-15 or IL15-RTXscFv-41BB-ligand were analyzed for FcγRIIIa (CD16a) expression by flow cytometry. Results from three NK cell donors are shown. [Modes for carrying out the invention] [Examples]

[0197] The present invention will be explained by examples.

[0198] Example 1 - Materials and Method cell culture The cell line was exposed to 6% atmospheric CO2. 2 The cells were cultured in appropriate medium at 37°C. The cultures were divided 2-3 times per week to maintain an optimal density for continuous cell growth. Subculturing involved removing the medium and transferring cells from the previous culture to fresh medium. Adherent cultures were washed with PBS and then detached with accutase or trypsin-EDTA.

[0199] Chinese hamster ovary CHO-S cells (FreeStyle® CHO-S® cells; R800-07, Thermo Fisher Scientific, Dreieich, GER) were cultured in a horizontal shaking incubator in CD CHO medium (10743-011, Life Technologies, Carlsbad, USA) supplemented with 1% GlutaMax (Life Technologies). After transfection via large-scale electroporation, CHO-S cells were cultured in CD OptiCHO® (12681, Life Technologies) supplemented with 1% GlutaMax® (35050-038, Life Technologies), 1% PLURONIC® F-68 (24040-032, Life Technologies), and 1% HT-Supplement (11067-030, Life Technologies). 24 hours after transfection, 1 mM Na-butyrate (B5887, Sigma-Aldrich) was added to the cell culture, and 48 hours after transfection, daily feeding was initiated with a 3.5% feedstock (containing CHO CD Efficient Feed® A (A10234-01, Life Technologies), a 14% yeastolate stock solution (0.5% Difco® TC Yeastolate, UF (292804, Life Technologies) soluble in H2O), 3.5% GlutaMax® (72400-021, Life Technologies), and 12.4% glucose (Sigma-Aldrich)). Fusion protein-producing cells were cultured in a horizontal shaking incubator at 32°C with 6% CO2, 95% atmospheric moisture, as recommended by the manufacturer.

[0200] Mouse CTLL-2 (ATCC® TIB-214®, LGC Standards GmbH, Wesel, GER) was cultured in RPMI-1640 + GlutaMax®, 1 mM sodium pirubate (Sigma-Aldrich), 10% inactivated fetal bovine serum (FBS; 1270-106, Life Technologies), 10% rat T-STIM® (including con A culture supplement) (354115, Corning GmbH, Kaiserslautern, GER), 100 U / ml penicillin, and 100 μg / ml streptomycin (15140-122, Life Technologies). Cells grew to approximately 2 × 10⁶ cells. 5 Before reaching a density of cells / ml, the cells should be 1-2 × 10⁶ 4 Subculture until the inoculation density reaches viable cells / ml.

[0201] GRANTA-519 (mantle cell lymphoma; ACC342, DSMZ, Braunschweig, GER) was cultured in DMEM medium (41965-039, Life Technologies) supplemented with 10% inactivated FBS, 100 U / ml penicillin, and 100 μg / ml streptomycin.

[0202] L-363 (plasma cell leukemia; ACC49, DSMZ, Braunschweig, GER) was cultured in RPMI-1640 medium (11835-030, Life Technologies) supplemented with 10% inactivated FBS, 100 U / ml penicillin, and 100 μg / ml streptomycin.

[0203] B cells and NK cells were freshly prepared using the Human B Cell Isolation Kit II (130-091-151, Miltenyi Biotec, Bergisch Gladbach, GER) or the Human NK Cell Isolation Kit (130-092-657, Miltenyi Biotec). After isolation, B cells and NK cells were cultured for up to 4 weeks in NK MACS basal medium (Miltenyi Biotec) supplemented with 1% NK MACS supplement (Miltenyi Biotec), 5% inactivated AB serum (P30-2501, PAN-Biotech GmbH, Aidenbach, GER), and 0.78 nM IL-15 (Miltenyi Biotec) or 18.7 nM EFP.

[0204] Cloning and generation of recombinant fusion proteins To generate the fusion proteins RTX-CD137scFv-IL-15;RTX-IL-15;RTX-CD137scFv;Her2-CD137scFv-IL-15, each fusion protein is synthesized from its individual components (CD20 secretion leader; cDNA sequences encoding the constant regions of the immunoglobulin heavy and light chains from CD20-VH, CD20-VL, and the CD20 antibody rituximab; cDNA sequences encoding CH1, CL, the constant region of the human immunoglobulin heavy chain 1 and the constant region of the copper light chain; and CD137VL, CD137VH, and 4-1BB (CD137)). cDNA sequences encoding variable heavy and light chain constant regions that construct specific scFvs; cDNA sequences encoding the sushi domain, hIL-15, sushi domain and human interleukin 15; cDNA sequences encoding GS15, GS20, 15-amino acid mobile linker (G4S)3 and 20-amino acid mobile linker (G4S)4; cDNA sequences encoding myc tag, His tag, c-myc epitope and hexahistidine tag (Eurofins, Ebersberg, Germany) were cloned into the expression vector pSEC-tag2. The accuracy of the cloned sequences was confirmed by Sanger sequencing of the final construct. To generate the fusion proteins RTX-NKp46scFv-IL-15, RTX-NKG2DscFv-IL-15, RTX-CD137scFv-IL-2, RTX-CD137scFvdss-IL-15, CD19-CD137scFv-IL-15, and IL15-RTXscFv-41BB-ligands, complete constructs were newly synthesized and cloned into pCDNA3.1(+) (Thermofischer Scientific, Geneart).

[0205] CHO-S cells were thawed two weeks before transfection. 3 × 10⁶ cells were incubated in a shaking incubator. 6 These cells were maintained in culture at 2 × 10⁶ cells / ml, 6% CO₂, 95% atmospheric moisture, 37°C, and 125 rpm. The day before transfection, the cells were raised to 2 × 10⁶ cells. 6Cells were seeded at a rate of 8 × 10⁶ cells / ml. For the expression of the desired protein, electroporation was performed according to the manufacturer's recommendations using a MaxCyte Flow Electroporation® Unit STX, electroporation chamber OC-400, and the program "CHO-S Protein Expression" (MaxCyte Inc., Gaithersburg, USA), and the resulting endotoxin-free vector (120 μg / transfection - split into 60 μg light chain vector and 60 μg heavy chain vector), resulting in 8 × 10⁶ cells. 7 CHO-S cells (FreeStyle® CHO-S® cells; R800-07 Thermo Fisher Scientific, Dreieich, GER) were transfected 10 times. The resulting fusion proteins containing the Fab fragment were purified from the cell culture supernatant using a gravity flow column (Bio-Rad Laboratories, Hercules, CA, USA) with CaptureSelect® IgG-CH1 affinity matrix (Thermo Fisher Scientific) and affinity chromatography, as recommended by the manufacturer. To purify the IL15-RTXscFv-41BB-ligand fusion protein, the supernatant was dialyzed three times against a wash buffer (50 mM NaH2PO4, 300 mM NaCl, 10 mM imidazole, pH 8), and then captured by Ni-NTA affinity chromatography (Qiagen), as recommended by the manufacturer. The protein was eluted using an elution buffer (50 mM NaH2PO4, 300 mM NaCl, 250 mM imidazole, pH 8). The protein was then dialyzed three times against PBS. To eliminate potential contaminants in aggregates, size exclusion chromatography was performed using an AEKTApure liquid chromatography system (Cytiva Europe GmbH, Freiburg im Breisgau, GER) according to the prescribed method. Protein concentrations were determined using the Pierce® BCA protein assay (Thermo Fisher Scientific) according to the manufacturer's protocol.

[0206] Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and gel filtration chromatography To determine the size and purity of the proteins, sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) was performed. For sample preparation, 5× loading buffer (either reducing or non-reducing) was added to each 3 μg protein sample, and the samples were heated to 95 °C for 10 min to denature the proteins. After cooling, the samples were applied to an SDS-bis-tris-polyacrylamide gel (10%, #456-1083, Bio-Rad Laboratories GmbH, Feldkirchen, GER). The proteins were separated at 120 V for 60 min according to their molecular mass. The gels were stained overnight with constant shaking in Coomassie Brilliant Blue solution (Carl Roth GmbH, Karlsruhe, Germany).

[0207] MTT proliferation assay To test the functionality of the fusion IL-15 component of the fusion protein, a colorimetric assay (MTT-based; 11465007001, Cell Proliferation Kit I, Roche, Mannheim, GER) was used to quantify metabolic cell activity. The MTT assay was performed using IL-2-dependent CTLL-2 cells. Due to the fact that IL-15 binds to the same receptor complex called IL-2R / IL-15Rβ and γC, CTLL-2 cells respond to IL-15 stimulation. After starvation of CTLL-2 cells (5 h, without addition of IL-2 (T-STIM)), 3×10 4 cells were transferred to a 96-well plate and incubated at 37 °C and 6% CO2 for 48 h. To test the functionality of various proteins, these proteins and recombinant human IL-15 were added in an equimolar concentration range of 62.6 nM to 0.61 pM. The absorbance of the dissolved formazan was measured spectrophotometrically at 570 nm using a Sunrise plate reader (Tecan Group Ltd., Maennedorf, CH). Evaluation was performed by GraphPad PRISM 5.0.

[0208] Flow cytometry analysis All immunofluorescence analyses were performed using a Navios EX-flow cytometer (Navios, Beckman Coulter, Brea, CA, USA). 1 × 10⁻⁶ samples were analyzed for each sample. 4 Individual events were collected, and dead cells and cellular debris were excluded by using appropriate forward and side scattering gating. Cells stained only with the secondary antibody served as a negative control. Immunofluorescence measurements were performed at the corresponding wavelengths (FITC / PE). Final analysis was performed using Kaluza C analysis software and GraphPad Prism 4.0 software.

[0209] target cell binding To determine the Fab-mediated target cell binding of various proteins, two different cell lines were used (GRANTA-519 for CD20; SK-BR-3 for 4D5). Each cell line measured 3 × 10⁶ cells. 5 Cells were incubated on ice for 30 minutes in a 1 / 5 serial dilution with 4.85 μM fusion proteins of different concentrations, washed twice with 1000 μl of PBA buffer (PBS containing 1% BSA), and stained with FITC-conjugated anti-human copper antibody (50 μg / ml, Southern Biotech, Birmingham, USA) on ice for 30 minutes. After the second wash, flow cytometry analysis was performed to analyze the cells.

[0210] 4-1BB bond To confirm the 4-1BB-scFv binding of the fusion protein, CEM-CRRF cells were stimulated to express CD137 on their cell surface. Stimulation was performed for 16 hours using 1.33 μM ionomycin (I0634, Sigma-Aldrich) and 16.2 nM PMA (P1585, Sigma-Aldrich). Cells incubated under the same conditions but using DMSO instead of PMA and ionomycin served as negative controls. Each cell sample size was 3 × 10⁶. 5Cells were incubated on ice for 30 minutes in a 1 / 5 serial dilution with 4.85 μM fusion proteins of different concentrations. After washing twice with 1000 μl of PBA buffer, the cells were incubated on ice for 30 minutes with FITC-conjugated anti-human copper antibody (50 μg / ml, Southern Biotech, Birmingham, USA). After washing the cells twice, flow cytometry analysis was performed.

[0211] Analysis of cell surface markers Total 3 x 10 5 Individual cells were washed with PBA. Next, the cells were incubated with 5 μl of directly labeled primary antibody on ice in the dark for 30 minutes. Cells incubated with the corresponding IgG isotype control served as a negative control. After incubation, the cells were washed once with 3 ml of PBA and then resuspended in 500 μl of PBA. Immunofluorescence measurements were performed at the corresponding wavelengths (FITC / PE).

[0212] Preparation of PBMCs, isolation of NK cells and B cells, and validation. Citrate-buffered blood samples were obtained from healthy donors or patients. Peripheral blood mononuclear cells (PBMCs) were isolated by Ficoll-Paque® PLUS (Cytiva Europe) density centrifugation at 2500 rpm and room temperature for 20 minutes without braking. Isolation of NK cells, T cells, and B cells was performed using the corresponding isolation kits (Human NK Cell Isolation Kit, Human B Cell Isolation Kit II, panT Cell Isolation Kit; Miltenyi Biotec, Bergisch Gladbach, GER) as described by the manufacturers. Cell purity analysis was performed using cell samples collected before cell separation and cell samples collected after isolation by flow cytometry with appropriate B, T, and NK cell markers (CD56-APC[IM2474], CD3-ChromeOrange[B00068], CD16-FITC[B49215], CD19-PE[A86355], IgG1-APC[IM2475], IgG1-PE[A07796, A74764], IgG1-FITC[A07795], IgG1-ChromeOrange[A96415], Beckman Coulter GmbH, Krefeld, GER).

[0213] The fusion protein induced proliferation of NK cells from purified NK cells or patient-derived PBMCs. To determine the (tumor) cell-dependent proliferation of NK cells, isolated NK cells were co-cultured in human NK MACS medium (+1% NK MACS supplement, +5% AB serum) with autotarget cells (e.g., B cells) in a 2:1 ratio (initial condition 1.5 × 10⁻⁶). 6 ( / ml). NK cells cultured without target cells and NK cells alone served as negative controls. Purified NK cells grown using the NK cell proliferation and activation kit from Miltenyi (130-094-483, Miltenyi Biotec), as recommended by the manufacturer, served as controls. To determine the target cell-dependent proliferation of NK cells from isolated patient-derived PBMCs, 1.0 × 10⁶ cells were cultured in human NK MACS medium (+1% NK MACS supplement, +5% AB serum). 6Patient-derived PBMCs were incubated under initial conditions of / ml. PBMCs cultured without EFP supplementation served as a negative control.

[0214] Co-culture was performed in an incubator with 6% CO2, 95% atmospheric moisture, and 37°C. Different fusion proteins (18.7 nM) were added to the co-cultured cells. Four days after the start, fresh medium and fusion protein were added. From day 7 onward, fresh medium and fusion protein were added every 3-4 days (days 7, 11, 14, 18, 21, and 25). On these days, the total cell count was determined using trypan blue (T8154, Sigma-Aldrich) and a Neubauer counting chamber (0640010, Paul Marienfeld GmbH & Co KG, Lauda-Koenigshofen, GER), totaling 1 × 10⁶ cells. 6 The cells were reseeded individually. On day 0 and days 7-29, appropriate NK cell markers (CD56-APC[IM2474], CD3-ChromeOrange[B00068], CD16-FITC[B49215], CD19-PE[A86355]; CD11a-FITC[IM0860U], CD44-PE[A32537], CD69-PE[IM1943], NKG2D[A08934], DNAM-1-PE, NKp30-PE[IM3709], NKp44-PE[IM3710], NKp46-PE[IM3711]; IgG1-APC[IM2475], IgG1-PE[A07796, A74764], IgG1-FITC[A07795], IgG1-ChromeOrange[A96415], Beckman Cells were characterized by flow cytometry analysis using Coulter GmbH, Krefeld, GER.

[0215] Analysis of cell-mediated cytotoxicity To characterize the functionality of the proliferated activated NK cells, a 51-chromium release assay was performed in the presence and absence of therapeutic antibodies. The ability of the proliferated activated NK cells to induce ADCC using effector cells was standard. 51This was performed by inducing Cr release. Proliferating NK cells were subjected to various effector cell-to-target cell (E:T) ratios (1:1, 1:2.5, 1:5, 1:10, and 1:20). 51 Target cells incubated with Cr were co-cultured. CD20-expressing cells (either autologous B cells from a healthy donor or the CD20-expressing cell line GRANTA-519) or autologous CD138 cells from a patient. + Multiple myeloma cells were used as target cells. 51 The cells were incubated with Cr for 2 hours. The monoclonal antibodies rituximab (RTX, 1 μg / ml; Roche, Basel, CH), elotuzumab (SLAMF7, 2 μg / ml; BMS, New York, USA), and daratumumab (CD38, 2 μg / ml; Janssen-Cilag GmbH, Neuss) from the patient's own environment were added. In all environments, trastuzumab (4D5, 1 μg / ml; Roche, Basel, CH) served as a negative control. The co-cultures were incubated at 37°C for 4 hours. Background lysis by only the proliferated activated NK cells was detected. Maximum lysis was detected by adding 1% Triton-X. Measurement was performed using a MicroBeta Trilux 1450 LSC & luminescence counter (Perkin Elmer, Waltham, USA).

[0216] Data processing and statistical analysis Graph and statistical analyses were performed using GraphPad PRISM 4.0 (GraphPad Software Inc., San Diego, CA). Experiments were always conducted under the same conditions. All data were analyzed and displayed in the same manner: all histograms were expressed as the mean ± standard error (SEM) of at least three biological copies, and statistical descriptions were performed by two-way analysis of variance (ANOVA) and Bonferroni post-hoc tests. The null hypothesis was rejected when p < 0.05.

[0217] Example 2 - Results of CD20 as a target on B cells Production and Purification Transpresentation of IL-15 and 4-1BB ligands in genetically modified target cells such as K562 or in bead-based systems enables ex vivo proliferation of NK cells for cell therapy. To eliminate the need for genetically modified feeder cells or bead-based systems, we designed a novel multifunctional fusion protein that enables the proliferation of specific NK cells and / or T cells. To deliver IL-15 and 4-1BB signaling in trans to NK cells or T cells / NKT cells, we created the fusion protein RTX-CD137scFv-IL-15, consisting of a Fab fragment of rituximab, the agonist scFv for 4-1BB, the sushi domain of the IL-15 receptor, and interleukin 15 (IL-15) (Figure 1, A). This protein is designed to bind to CD20 on autologous B cells, thereby enabling trans induction of the IL-15 receptor and 4-1BB on NK cells or T cells / NKT cells. To investigate the contribution of individual molecular components to NK cell activation and proliferation, we created additional protein variants of RTX-CD137scFv-IL-15, RTX-IL-15;RTX-CD137scFv;Her2-CD137scFv-IL-15, which lack specific components of RTX-CD137scFv-IL-15.

[0218] To generate the protein, CHO-S cells were simultaneously introduced with the expression vectors encoding the heavy chain derivative and their respective light chains. Purification of the fusion protein from the culture supernatant was performed by affinity chromatography. Quantitative size exclusion chromatography was performed to remove residual contaminants and the possibility of polymers or aggregates. The purity and molecular weight of the isolated protein were analyzed by SDS-PAGE and Coomassie blue staining (Figure 1, C). Under reducing conditions, the light chain (LC) appeared with a calculated molecular weight of 25.7 kDa, and the heavy chain derivative (HC) appeared with a molecular weight of 52.6–79.3 kDa (RTX-CD137scFv-IL-15(79.3 kDa); RTX-IL-15(52.6 kDa); RTX-CD137scFv(55.7 kDa); Her2-CD137scFv-IL-15(79.3 kDa)). Similar analyses using non-reducing conditions revealed the integrity of each protein, with molecular masses being RTX-CD137scFv-IL-15(105kDa); RTX-IL-15(78.3kDa); RTX-CD137scFv(81.4kDa); and Her2-CD137scFv-IL-15(105kDa).

[0219] antigen binding Flow cytometry analysis was performed to determine the binding ability of the fusion protein. Either Granta-519 cells (for CD20 binding; Figure 1, D) or 4-1BB (CD137)-positive stimulated CRF-CEM cells (for 4-1BB binding; Figure 1, E) were used.

[0220] CD20-specific fusion protein (RTX-CD137scFv-IL-15- [red filled circle], EC 50 Value 231nM; RTX-CD137scFv - Blue filled square, EC 50 Value 205nM; RTX-IL-15 - light purple filled square, EC 50The binding ability at a value of 414 nM did not show a significant difference. For RTX-IL-15, a significant difference was observed at only one concentration compared to RTX-CD137scFv-IL-15 and RTX-CD137scFv (Figure 1, D). As expected, binding was not observed in Her2-negative Granta-519 cells for trastuzumab (black filled triangle) and Her2-specific control fusion protein (Her2-CD137scFv-IL-15-black circle).

[0221] CD20-specific fusion protein containing 4-1BB-specific scFv (RTX-CD137scFv-IL-15-red filled circle, EC 50 Value 139nM; RTX-CD137scFv - Blue filled square, EC 50 The value 81 nM did not show a significant difference in binding to CD137-positive CRRF-CEM cells. CD20-specific fusion protein containing 4-1BB-scFv (RTX-CD137scFv-IL-15-red filled circle; RTX-CD137scFv-blue filled square) and Her2-specific fusion protein (Her2-CD137scFv-IL-15-black circle, EC) 50 A significant difference was observed between the values ​​(511 nM) and the above. This suggests that the specific Fab fragment used to design the fusion protein may affect the overall activity of the protein. CD137 binding was not observed for the fusion protein lacking 4-1BB-specific scFv (RTX-IL-15 - light purple filled square) and trastuzumab (black filled triangle) (Figure 1, E).

[0222] Functional activity of IL-15 component To determine the functionality of the IL-15 component of the fusion protein, a cellular metabolic activity assay was performed using IL-15-responsive mouse CTLL-2 cells (Figure 1, F). To compare the stimulatory activity of various fusion proteins, serial dilutions of the fusion protein (RTX-CD137scFv-IL-15 - red filled circle; RTX-CD137scFv - blue filled square; RTX-IL-15 - light purple filled square; HER2-CD137scFv-IL-15 - black circle) and recombinant human interleukin 15 (hIL-15, IL-15 - black triangle) were prepared and applied in equimolar amounts. hIL-15 served as a positive control. Relative metabolic activity (%) was plotted against protein concentration (pM). The highest hIL-15 value was set to 100%, and the EC50 value was calculated. A significant difference was observed between recombinant hIL-15 and the fusion protein. The recombinant IL-15 EC50 value was 0.014 nM, but the IL-15-containing fusion proteins (RTX-CD137scFv-IL-15 - red filled circle; RTX-IL-15 - light purple filled square; HER2-CD137scFv-IL-15 - black circle) showed EC50 values ​​between 9 nM (RTX-IL-15) and 14 nM (HER2-CD137scFv-IL-15 - black circle; Figure 1, F). The IL-15-containing fusion proteins (RTX-CD137scFv-IL-15 - red filled circle; RTX-IL-15 - light purple filled square; HER2CD137scFv-IL-15 - black circle) did not show a significant difference in activity. As expected, no stimuli activity was observed for the fusion protein lacking the IL-15 component (RTX-CD137scFv - blue filled square; Figure 1, F).

[0223] NK cell proliferation To determine the ability of a novel fusion protein (RTX-CD137scFv-IL-15) containing all structural components to induce NK cell proliferation, newly isolated NK cells were incubated with the fusion protein in the presence of autologous B cells. The fusion protein strongly induced NK cell proliferation. Proliferation rates between 10x and 10,000x were observed after 28 days (Figure 2, A). Only one donor showed a proliferation rate of less than 10x (8.33x), and one donor showed a proliferation rate of less than 100x (52.05x), but the majority of donors showed proliferation rates between 100x and 10,000x (11 donors between 100x and 1,000x, and 6 donors between 1,000x and 10,000x). Compared to commercially available bead-based proliferation systems, the IL-15-based fusion protein showed a slightly improved proliferation rate, but the difference was not statistically significant (Figure 2, B). In the following series of experiments, we analyzed the requirements of individual structural components and the need to provide IL-15 / 4-1BB signaling through transpresentation (by opsonization of B cells) to induce NK cell proliferation (Figure 2, C). The experiments demonstrated that fusion proteins possessing all structural components, in particular, could induce potent NK cell proliferation. Significantly lower proliferation rates were observed in the absence of B cells. This indicated that transpresentation is necessary to provide optimal IL-15 / 4-1BB signaling.

[0224] Activation Analysis The cytotoxic activity of NK cells is regulated by a set of receptors that recognize the absence of self-proteins and the presence of stress ligands on target cells. NK cell activation is characterized by increased expression of specific NK cell surface receptors. Flow cytometry-based analysis was performed to determine the state of NK cells proliferated with recombinant fusion proteins. NK cells express different amounts of the Fc receptor FcγRIIIa (CD16) depending on their activation state. To evaluate this expression, flow cytometry analysis was performed on days 0 and 7 using commercially available CD16, CD56, and CD3 antibodies. This showed that proliferation with the novel fusion protein resulted in higher levels of CD56 compared to proliferation using a commercially available bead-based proliferation system (54%; Figure 3, A). + CD16 + This study demonstrates the production of NK cells (76%). The expression of other NK cell markers was determined on days 0 and 28 of culture (Figure 3, B). By comparing receptor expression at the start of proliferation with that of NK cells that had undergone 28 days of proliferation, it can be shown that the expression of selected NK cell markers was significantly increased (Figure 3, B). Data represent the mean of three independent measurements, and error bars represent ±SEM. * =<0.05.

[0225] Proliferative cytotoxicity of NK cells To determine the cytolytic capacity of proliferating NK cells, NK cell-mediated tumor cell lysis was measured by performing cytotoxic assays using different target cells. Positive lysis of K562 cells was demonstrated with NK cells proliferated using the novel fusion protein of the present invention at various effector-to-target ratios (Figure 4A). Significant lysis exceeding 30% was already observed at a low E:T ratio of 1:1. In the following series of experiments, lysis was performed on a panel of tumor cells representing various tumor entities. Significant lysis was observed in all tumor cell lines tested (Figure 4B). The degree of lysis ranged from 20% to 75%. To test whether proliferating NK cells are still physiologically regulated and therefore do not attack non-malignant target cells, cytotoxic assays were performed using autologous non-malignant B cells as target cells. No significant lysis was observed when non-malignant B cells were used as target cells. This indicates that highly activated proliferating NK cells are still physiologically regulated (Figure 4B).

[0226] The experiment was conducted with a fixed E:T ratio of 10:1. The presented data are the mean + / - SEM values ​​from three NK cell donors.

[0227] ADCC mediated by proliferating NK cells In addition to innate cytotoxicity, NK cells can induce antibody-dependent cell-mediated cytotoxicity (ADCC) through FcγRIIIa binding. As demonstrated above, NK cells proliferated with the novel fusion protein of the present invention expressed high levels of FcγRIIIa on the majority of cells (>75%). Different tumor cell lines of B cell lineages were used to test NK cell-mediated ADCC. Proliferating NK cells demonstrated significant lysis of all tumor cell lines tested, and the lysis rate was increased by adding a tumor-targeting monoclonal antibody (rituximab, CD19-DE) at a fixed E:T ratio (Figure 5, A+B). In the following series of experiments, NK cell-mediated ADCC using allogeneic NK cells at various E:T ratios was analyzed (Figure 5, C, left panel). In this case as well, proliferating NK cells significantly lysed target cells at various E:T ratios in the absence of the therapeutic antibody. The lysis of tumor cells was enhanced by the addition of the therapeutic antibody rituximab. Next, similar experiments were performed using autologous non-malignant B cells as target cells. Importantly, proliferating NK cells were unable to induce lysis of non-malignant cells with a high E:T ratio (Figure 5, C, right panel). This highlights that highly activated proliferating NK cells remain physiologically controlled and do not attack non-malignant cells. Lysis of non-malignant B cells can be achieved by adding the therapeutic antibody rituximab. Under similar conditions, NK cells proliferated with the multifunctional fusion protein of the present invention were compared with NK cells proliferated using a commercially available bead-based proliferation system. No significant difference was observed in the innate cytotoxicity and ADCC of non-malignant B cells, but the proliferating NK cells of the present invention more potently induced ADCC against tumor cells (Figure 5, D).

[0228] Proliferation and cytotoxic activity of NK cells from multiple myeloma patients In the following series of experiments, we tested whether the novel fusion protein of the present invention could induce the proliferation of NK cells from tumor patients. Mononuclear cells were used from peripheral blood or bone marrow aspirate from multiple myeloma patients. In these initial experiments, proliferation rates between 10- and 400-fold were achieved (Figure 6, A). When the control molecule was used, no or very little proliferation was observed. This further demonstrates that all structural components in the molecule are necessary to induce optimal NK cell proliferation. Under these experimental conditions, NK cells proliferated preferentially. By day 16, 85% of the culture already consisted of NK cells expressing high levels of FcγRIIIa (Figure 6, B). Finally, primary tumor cells were tested with a cytotoxic assay. Primary myeloma cells were significantly lysed by allogeneic NK cells (Figure 6, C, left panel). The killing of tumor cells was significantly enhanced by the addition of the therapeutic antibody elotuzumab. In the native environment, tumor cell killing was observed only in the presence of the therapeutic antibodies daratumumab or elotuzumab. This indicates that these tumor cells still provide a strong signal to evade innate cytotoxicity.

[0229] Example 3 - Results of BCMA as a target on B-cell malignancies To assess the broad applicability of the proposed concept, a second target structure was evaluated using BCMA. Since antibody titer is a crucial parameter for the activity of many antibody derivatives, an additional construct with two Fab fragments (DuoFab) as the target domain was designed.

[0230] Design and expression of fusion proteins with bivalent target antigen binding ability. Four constructs (RTX-DuoFab-CD137scFv-IL-15, RTX-CD137scFv-IL-15, BCMA-DuoFab-CD137scFv-IL-15, BCMA-CD137scFv-IL-15, 7A) were generated in CHO-S cells by transient transfection and purified by affinity chromatography. Macromers and aggregates were removed by size exclusion chromatography (Figure 7, B).

[0231] Biochemical characterization of fusion proteins with bivalent target antigen binding ability The purified DuoFab-based proteins were further analyzed by SDS-PAGE and Coomassie blue staining or Western blotting. Under non-reducing conditions, the molecules exhibited the expected molecular weight of 150 kDa with no signs of degradation. Under reducing conditions, the molecules separated into light and heavy chain derivatives. The identity of each polypeptide chain was confirmed by Western blotting using copper light chain or polyhistidine-specific antibodies (Figure 8).

[0232] Binding properties of fusion proteins with bivalent target antigen binding ability Flow cytometry analysis was performed to determine the binding ability of the antibody fragments. Either CD20-positive Granta-519 cells (Figure 9, A) or Lenti-X cells transfected with an expression vector encoding BCMA cDNA were used (Figure 10, B). In CD20 binding analysis, RTX-DuoFab-CD137scFv-IL-15 and RTX-CD137scFv-IL-15 were compared, while BCMA-DuoFab-CD137scFv-IL-15 and BCMA-CD137scF-IL-15 served as negative controls. In BCMA-binding analysis, BCMA-DuoFab-CD137scFv-IL-15 and BCMA-CD137scFv-IL-15 were compared, and RTX-DuoFab-CD137scFv-IL-15 and RTX-CD137scFv-IL-15 served as negative controls.

[0233] We analyzed dose-dependent binding and EC 50The values ​​and Kd values ​​were calculated. The highest determined relative mean fluorescence intensity (t0 value for cell surface retention) was set to 100%, and all other values ​​were normalized to this point. The fitted determined relative mean fluorescence intensity (rel.MFI, %) was plotted against protein concentration (nM). Dose-response was plotted as dose-response curves and hyperbolas for CD20 binding analysis (Figure 9, B+C), and as a hyperbolas only for BCMA binding analysis (10, C). In the cell surface retention assay, dissociated molecules were removed from the supernatant at various time points. The remaining cell surface-bound molecules were determined by flow cytometry (Figure 9, D and 10, D).

[0234] Both dose-response curves and hyperbolic analyses showed significant differences in binding ability between RTX-DuoFab-CD137scFv-IL-15 and the RTX-CD137scFv-IL-15 construct. RTX-DuoFab-CD137scFv-IL-15 (EC50: 278.4nM) had a 2.7 times lower EC50 compared to RTX-CD137scFv-IL-15 (EC50: 740.7nM). 50 The values ​​were shown (Figure 9, B). This result was confirmed by hyperbolic analysis, making it possible to calculate the Kd values ​​for each. RTX-DuoFab-CD137scFv-IL-15 (Kd 47.9nM) showed a Kd value 2.9 times lower than RTX-CD137scFv-IL-15 (Kd: 138.9nM, Figure 9, C). The cell surface retention assay reflected previous findings. 30 minutes from the baseline, only 50% of the rel.MFI was observed in RTX-CD137scFv-IL-15. In contrast, the rel.MFI of RTX-DuoFab-CD137scFv-IL-15 remained above 50% even after 180 minutes (Figure 9, D).

[0235] Similar results were obtained in the second model system. Binding analysis plots showed a significant difference in binding ability between BCMA-DuoFab-CD137scFv-IL-15 and BCMA-CD137scFv-IL-15. BCMA-DuoFab-CD137scFv-IL-15 (Kd: 56.5 nM) showed a 16.4-fold lower Kd value compared to BCMA-CD137scFv-IL-15 (Kd: 924.5 nM, Figure 10C). Preliminary cell surface retention assays confirmed previous findings obtained with CD20-specific molecules (Figure 10, D).

[0236] In summary, all generated antibody derivatives can be shown to bind specifically to their respective target cells / target antigens. Furthermore, both antibody derivatives containing the two Fab fragments exhibit significantly stronger binding ability compared to monovalent antibody derivatives. As a result, surface retention of DuoFab-based molecules is improved, which may be a desirable property, especially for in vivo applications.

[0237] Antibody derivatives with monovalent and bivalent tumor cell-binding domains show no difference in CD137 binding. The antibody derivatives were designed to bind to CD137 on activated NK cells via the CD137-scFv fragment. Following stimulation with PMA and ionomycin, CCRF-CEM cells expressed CD137 on their cell surface. Unstimulated cells served as negative controls. Antigen expression on corresponding cells was confirmed before commencing binding analysis (Figure 11, A+B). Dose-dependent binding was analyzed to compare different antibody derivatives, and EC was performed. 50The values ​​were calculated. The highest determined relative mean fluorescence intensity was set to 100%, and all other values ​​were normalized to this point. The fitted determined relative mean fluorescence intensity (rel.MFI, %) was plotted against protein concentration (nM) (Figure 11, C). Analysis confirmed that the antigen CD137 is expressed only in stimulated CCRF-CEM cells (Figure 11, B). In unstimulated CCRF-CEM cells, no significant difference in MFI was observed compared to isotype controls (Figure 11, A). Stimulated CCRF-CEM cells were confirmed to be CD137-positive. The affinity of the different antibody variants did not show significant differences. All four antibody derivatives showed nearly the same binding ability to stimulated CCRF-CEM cells. As expected, with the exception of BCMA-DuoFab-CD137scFv-IL-15, the antibody derivatives did not bind to CD137-unstimulated CCRF-CEM cells. In summary, all antibody derivatives analyzed bound to the antigen CD137, and therefore, it was demonstrated that they contained functionally active CD137-scFv.

[0238] There is no difference in IL-15-dependent stimulating activity among different antibody derivatives. To determine the IL-15 functionality of antibody derivatives, a cellular metabolic activity assay was performed using CTLL-2 cells. CTLL-2 is a mouse cell line that is IL-2 dependent and responds to IL-15 stimulation. To compare the stimulating activity of different antibody derivatives, serial dilutions of antibody derivatives and recombinant human interleukin 15 (hIL-15) were prepared and applied at equimolar concentrations. hIL-15 served as a positive control. Relative metabolic activity (%) was plotted against protein concentration (pM) (Figure 12). The highest hIL-15 value was set to 100%, and EC was used. 50 The value was calculated.

[0239] IL-15-dependent cell activity showed a significant difference between recombinant IL-15 and IL-15-based antibody derivatives. EC of recombinant IL-15 50 The value was 2.28 pM, but the antibody derivative had an EC of between 11,640 pM and 17,942 pM. 50The values ​​are shown (Figure 12). Therefore, recombinant IL-15 showed 6,612 times higher activity compared to the antibody derivative. As shown in Figure 12, the series of antibody derivatives did not show significant differences in CTLL-2 stimulation.

[0240] There is no difference in the ability of MonoFab and DuoFab derivatives to induce NK cell proliferation. In the following series of experiments, different molecules with monovalent or bivalent target antigen binding ability were compared in terms of NK cell proliferation (Figure 13). No significant difference was observed in their ability to induce NK cell proliferation. This indicates that bivalent target antigen binding does not significantly affect this property ex vivo (Figure 13).

[0241] NK cells proliferated with antibody derivatives exhibited an activated phenotype. NK cell activation is regulated by a set of receptors that recognize the absence of self-proteins and the presence of stress ligands on target cells. NK cell activation is characterized by increased expression of specific NK cell markers. Typically, these markers include the antigen CD69 and the hyaluronic acid receptor CD44, as well as NKp44. Other activating receptors are NKp30, NKp46, CD16a, DNAM-1, and NKG2D. In tumor and chronic infection environments, NK cells can exhibit an exhausted phenotype (Gardiner, 2017). Phenotypic alterations are characterized by downregulation of specific receptors such as NKG2D, CD16a, NKp30, NKp44, and NKp46. Phenotypic alterations are associated with reduced effector function and therefore with poor control of malignant tumors or infections. Flow cytometry-based analysis was performed to determine the state of NK cells proliferated with antibody derivatives. Expression of different NK cell markers was determined at the start and end of the NK cell proliferative phase (Figure 14). The expression of NK cell markers was considered synonymous with fluorescence intensity. By comparing receptor expression at the start of proliferation with that of NK cells that had undergone 28 days of proliferation, it was shown that the expression of specific NK cell markers showed a significant increase (Figure 14).

[0242] In NK cells proliferated with the RTX-CD137scFv-IL-15 construct, NK cell markers CD44, NKp44, CD69, and DNAM-1 showed significantly increased expression levels. On day 0, hyaluronic acid receptor CD44 expression showed a fluorescence intensity of 167.9 MFI. By the end of proliferation, expression had increased to 645.7 MFI. NKp44 expression increased 12-fold. Similarly, the expression of CD69 and DNAX accessory molecule-1 (DNAM-1) also increased significantly. All other receptors analyzed also showed increased surface expression, but the differences were not statistically significant. Similar results were obtained for NK cells proliferated with the RTX-DuoFab-CD137scFv-IL-15 molecule.

[0243] Determination of NK cell marker expression in NK cells incubated with BCMA antibody derivatives revealed different expression patterns. Only NKp44 and CD69 showed significant differences among all tested samples. No differences were observed between the DuoFab-based and MonoFab-based constructs.

[0244] NK cells proliferated using DuoFab-based fusion proteins are not cytotoxic to non-malignant B cells but mediate ADCC. Since proliferating NK cells exhibit an activated phenotype, it is important to analyze whether these cells exhibit cytotoxic activity against non-malignant cells. Non-malignant autologous B cells were used as target cells in the chromium release assay. Even at a high E:T ratio of 20:1, no lysis of non-malignant B cells was observed (Figure 15). Opsonization of non-malignant B cells with the CD20-specific antibody rituximab induced significant lysis (Figure 15). These data demonstrate that NK cells proliferated by bivalent binding fusion proteins remain physiologically regulated and capable of distinguishing between non-malignant and malignant tissues, similar to the results obtained with monovalent target fusion proteins. These inhibitory self-recognition signals can be overcome by potent activating signals, such as FcγRIIIa-induced signals by the antibody Fc domain.

[0245] NK cells proliferated with DuoFab-based fusion proteins are cytotoxic to lymphoma cells, and this cytotoxic activity can be enhanced by combining them with monoclonal antibodies. In contrast to results obtained using non-malignant B cells as target cells, lymphoma cells (GRANTA-519 cells) were significantly lysed by proliferating NK cells (Figures 16 and 17). No significant difference was observed between NK cells proliferated with monovalent or bivalent fusion proteins. The cytotoxic capacity of NK cells was further enhanced by the addition of therapeutic antibodies such as rituximab (Figures 16 and 17).

[0246] The frequency of B cell / target cell-mediated NK cell stimulation and the NK cell-to-target cell ratio have an effect on NK cell proliferation. To further optimize the proliferation procedure, we analyzed two additional parameters that may influence the magnitude of NK cell proliferation. In the first series of experiments, we analyzed the frequency of NK cell stimulation by target cells (Figure 18). Two stimulations appeared optimal for monovalent target molecules, while for bivalent target molecules, four stimulations resulted in maximum proliferation in two out of three donors. For both types of molecules, a 4:1 NK cell:target cell ratio was optimal to achieve maximum NK cell proliferation (Figure 19).

[0247] summary Using the BCMA fusion protein, the concept of ex vivo / in vivo targeted proliferation of NK cells using the fusion protein of the present invention was demonstrated to be broadly applicable to various target structures on NK target cells. Alternative molecular design for bivalent binding to target cells was successfully achieved by fusion of a second Fab fragment. No significant differences were observed between monovalent and bivalent target molecules regarding NK cell proliferation and cytotoxic activity of proliferating NK cells. The main functional differences between these molecules were observed in surface retention assays. The bivalent target molecule exhibited long-term surface retention, a property favorable for in vivo application.

[0248] Example 4 - T cell proliferation IL-15 receptor and CD137 are also expressed by different subsets of (activated) T cells. Thus, the novel fusion proteins of the present invention may also be suitable for T cell proliferation ex vivo and in vivo. In a first series of experiments, isolated MNC (Figure 20, A) or purified T and B cell co-cultures (Figure 20, B) were incubated in the presence of different fusion proteins containing either all the structural components necessary to grow NK cells or derivatives lacking one of the important components. Similar to the results observed in NK cells, T cells also significantly proliferated, and this effect was most prominent with constructs containing all the structural components. Interestingly, CD3+ / CD16+ T cells also significantly proliferated. These cells are likely NKT and / or γδ T cells. Taken together, these data clearly demonstrate that the novel fusion proteins of the present invention potently induce the proliferation not only of NK cells but also of the CD3-positive T cell population.

[0249] In the next experiment, purified T cells (pan T cell isolation kit, Miltenyi Biotec) and B cells isolated from the same donor were co-cultured in the presence of RTX-CD137scFv-IL-15 according to the protocol used for NK cell proliferation. On days 0, 14, and 21, the content of vδ1 and vδ2 γδ T cells was analyzed by multicolor flow cytometry (Figure 21A / B). For both donors analyzed, significant proliferation of both vδ1 and vδ2 γδ T cells was observed. Proliferation of up to 1239-fold for vδ1 T cells and 546-fold for vδ2 γδ T cells was measured. Taken together, these data demonstrate that RTX-CD137scFv-IL-15 can induce significant proliferation of γδ T cells, which are also attractive immune effector cells for developing immunotherapy.

[0250] Example 5 - Proliferation of cytokine-induced memory-like NK cells Memory-like NK cells represent an interesting population of immune effector cells for therapeutic applications due to their increased cytolytic capacity. Overnight culture of NK cells in IL-12, IL-15, and IL-18 polarizes NK cells into a memory-like phenotype (Romee, Blood, 2012; Romee, Sci Transl Med, 2016). Here, it was tested whether NK cells polarized into a memory-like phenotype could be expanded by the novel fusion protein RTX-CD137scFv-IL-15 of the present invention. Expansion by RTX-CD137scFv-IL-15 as a single agent in the presence of B cells as described above (Figure 22A), expansion phase by RTX-CD137scFv-IL-15 in the presence of B cells after overnight culture of NK cells with IL-12, IL-15, and IL-18 (Figure 22B), and expansion phase by RTX-CD137scFv-IL-15 in the presence of B cells after overnight culture of NK cells with IL-12, IL-18, and the expansion molecule RTX-CD137scFv-IL-15 (Figure 22C) were compared among three different assay conditions. NK cells were evaluated for expression of FcγRIIIa (CD16a) by flow cytometry using commercially available CD16 and CD56 antibodies before, during, and after the expansion phase (day 0, day 14, and day 21). Data show representative results. Assay conditions B and C resulted in a higher percentage of CD56 / CD16 double-positive cells (>90%) compared to NK cells (>70%) that received only expansion by RTX-CD137scFv-IL-15 as a single agent.

[0251] Example 6 - Design of alternative molecular variants and comparison with fusion proteins based on the natural 4-1BB ligand (closest competing molecular design) To further investigate the potential of prototype molecular design and to gain deeper insights into the relative contributions of individual molecular components to NK cell proliferation, alternative molecular designs were evaluated (Figure 23). A) A disulfide-stabilized version of RTX-CD137scFv-IL-15 was designed to reduce protein multimer formation during synthesis. B) Protein variants containing scFv fragments that bind to alternative costimulatory receptors (NKG2D, NKp46) were designed to evaluate the effects of costimulatory changes mediated by alternative activating receptors on NK cells. C) Molecules addressing an alternative target structure (CD19) on B cells were designed to further analyze the effects of the target structure on B cells on NK cell proliferation. D) IL-15 was replaced with IL-2 to evaluate the effects of using alternative cytokines in molecular design. E) The closest competing molecule design (IL15-RTXscFv-41BB-ligand) using a native ligand (4-1BB ligand) and a B cell target scFv derived from a Fab fragment (rituximab) for stimulating CD137, as used in the design of the present invention, was evaluated for its ability to promote NK cell proliferation. The molecular structure and linker sequence of IL15-RTXscFv-41BB-ligand were designed according to Kermer et al. (Mol Cancer Ther, 2014).

[0252] All molecules were expressed in CHO-S cells by transient transfection as described above. Fab-containing molecular types were purified by CH1-specific affinity chromatography, and the IL15-RTXscFv-41BB-ligand molecule was purified by Ni-NTA chromatography using a standard procedure with an integrated His-Tag. After extensive dialysis against PBS, all proteins were subjected to preparative size exclusion chromatography to remove aggregates. Following size exclusion chromatography, proteins were quantified by BCA and analyzed by SDS-PAGE and Coomassi staining under reducing or non-reducing conditions. All proteins exhibited the expected migration characteristics based on the calculated protein molecular mass. For its molecular design, the Fab-based fusion protein showed two protein bands representing the light chain and each heavy chain derivative under reducing conditions (Figure 24A). No signs of degradation or contaminants were observed, confirming the accurate generation of the molecules. Evaluation under non-reducing conditions showed the accurate assembly of the Fab-based double-stranded molecules (Figure 24B, lanes 1-7). The IL15-RTXscFv-41BB-ligand molecule consists of a non-covalent trimer (a collection of 4-1BB ligands), and therefore, monomeric molecular weight transfer is expected in SDS-PAGE (Figure 24B, lane 7). The exact assembly of this trimer protein was confirmed by size exclusion chromatography (data not shown).

[0253] In the first series of experiments, using the assay settings described above, RTX-CD137scFv-IL-15 and IL15-RTXscFv-41BB-ligand were compared for their ability to proliferate purified NK cells. After 14 days, RTX-CD137scFv-IL-15 showed a 98.5-fold proliferation, while IL15-RTXscFv-41BB-ligand showed a 26.4-fold proliferation (Figure 25). These data demonstrate that the molecular design of RTX-CD137scFv-IL-15, and possibly the use of the agonist CD137-scFv instead of the natural 4-1BB ligand, resulted in significantly improved proliferation rates despite inducing the same receptor on B cells and NK cells. To investigate the dynamics of proliferation, NK cells were labeled with CFSE (carboxyfluorescein succinimimidyl ester; CellTrace® CFSE cell proliferation kit) and left untreated (NK cells only), co-incubated with B cells (NK cells + B cells), or co-incubated with B cells in the presence of RTX-CD137scFv-IL-15 (NK cells + B cells + RTX-CD137scFv-IL-15). After each cell division, the CFSE signal was reduced / diluted to allow for accurate measurement of cell division. After 5 days, CFSE was measured by flow cytometry to calculate cell division. Cells that underwent at least one cell division in 5 days were considered proliferative. Interestingly, approximately 80% of NK cells were proliferative on day 5 (Figure 25B). This feature, which induces a significant proportion of NK cells into proliferation, may explain the superior proliferative capacity of NK cells compared to IL15-RTXscFv-41BB-ligand. These findings are consistent with published data on the NK cell activation ability of Fap-directed fusion proteins with the same design as IL15-RTXscFv-41BB-ligand, which did not show a significant ability to induce NK cell proliferation on day 4 post-stimulation (Beha, Mol Cancer Ther, 2019). Interestingly, even an improved alternative molecular design using a single-chain 4-1BB trimer (type: IL15-scFv-sc4-1BB) induced proliferation in only about 15% of NK cells at high protein concentrations (Beha, Mol Cancer Ther, 2019).This potentially unexpected differential behavior of the novel fusion protein of the present invention compared to the published fusion protein may suggest that alternative molecular design and the use of the agonist CD137-scFv instead of the natural 4-1BB ligand provide qualitatively different co-stimulatory signals, resulting in a significantly improved ability to induce NK cell proliferation.

[0254] When IL-15 was replaced with IL-2 in the molecular design, the resulting fusion protein RTX-CD137scFv-IL-2 demonstrated nearly identical proliferation rates compared to RTX-CD137scFv-IL-15 (Figure 26). These data indicate that IL-2 can replace IL-15 in the molecular design of the present invention in terms of proliferation. Due to IL-2's inherent ability to efficiently activate Treg cells, this molecule may be of interest for potentially promoting the proliferation of regulatory T cells (Harris, Clin Exp Immunol, 2023).

[0255] In the fusion protein CD19-CD137scFv-IL-15, the v region of the CD20-Fab fragment was replaced with a v region possessing CD19 specificity. Interestingly, CD19-CD137scFv-IL-15 showed significantly reduced proliferative capacity compared to RTX-CD137scFv-IL-15. This suggests that the biology, epitope specificity and / or affinity of the target device, as well as the surface expression level of the addressed target antigen on B cells, play a crucial role in the proliferative range of NK cells (Figure 27).

[0256] The effects of the co-stimulatory signals provided by the proliferation molecules of the present invention were evaluated in more detail by replacing CD137-scFv with NKG2D-scFv or NKp46-scFv. All molecules induced significant proliferation of NK cells. Interestingly, CD137 co-stimulation resulted in a superior proliferation rate. This indicates that the quality of the co-stimulatory signal has a significant impact on the ability of molecules to induce NK cell proliferation.

[0257] Finally, we created a disulfide-stabilized molecular variant in which cysteine ​​was introduced into CD137-scFv within the consensus framework region (following Brinkmann, PNAS, 1993) and analyzed its tendency to form multimers / aggregates and its NK cell proliferation capacity. The disulfide bond-stabilized molecule RTX-CD137scFvdss-IL-15 showed a decrease in multimer levels after the first chromatographic step compared to RTX-CD137scFv-IL-15, while retaining full NK cell proliferation capacity. Therefore, this novel molecular design demonstrated improved production characteristics.

[0258] In summary, all novel molecular designs tested under the described assay conditions demonstrated improved NK cell proliferation characteristics compared to IL15-RTXscFv-4-1BB-ligand (Figure 30).

[0259] In the following series of experiments, the cytolytic ability of NK cells proliferated with various fusion proteins was analyzed in a classical chromium-releasing assay. K562 cells, which are highly sensitive to NK cell-mediated lysis, were potently killed by all proliferating NK cell preparations (Figure 31). Interestingly, when GRANTA-519 lymphoma cells, which are less sensitive to NK cell-mediated killing, were used as target cells, RTX-NKp46scFv-IL-15 proliferating NK cells were as effective in lysing the target cells as NK cells proliferated with the IL15-RTXscFv-41BB-ligand competing molecule. Importantly, all other molecules showed a superior increase in innate cytotoxicity compared to the IL15-RTXscFv-41BB-ligand (Figure 32). Most notably, all NK cell preparations grown with any of the novel proliferation molecules were significantly more effective in mediating ADCC compared to NK cells grown with the IL15-RTXscFv-41BB-ligand competing molecule (Figure 32, with antibody) (in the presence of rituximab, with antibody). In summary, these data demonstrate that the novel fusion protein of the present invention is more efficient than the IL15-RTXscFv-41BB-ligand in inducing NK cell proliferation, and that when combined with a monoclonal antibody (rituximab), the proliferated NK cells were effective in mediating innate cytotoxicity and ADCC.

[0260] To investigate the fundamental mechanisms that contribute to the difference in cytotoxic activity between the novel fusion protein and the IL15-RTXscFv-41BB-ligand competing molecule, we examined the expression of selected surface markers. Interestingly, increased expression of CD69, NKp30, and FcγRIIIa was observed (Figure 33). While elevated NKp30 levels may explain higher levels of intrinsic cytotoxicity, clearly, higher FcγRIIIa expression explains superior ADCC activity. Further analysis revealed that proliferation of NK cells induced by the novel fusion protein RTX-CD137scFv-IL-15 resulted in a higher proportion of FcγRIIIa-highly expressing NK cells compared to NK cells proliferated with the IL15-RTXscFv-41BB-ligand competing molecule (Figure 34).

[0261] In summary, these data demonstrate a clear difference in the degree of proliferation and cytolytic activity between the novel fusion proteins provided herein and conventional, technologically advanced IL15-RTXscFv-41BB-ligand competitive molecular designs based on natural 4-1BB ligands.

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Claims

1. (a) Antibodies or antibody fragments that bind to antigens expressed on the surface of target cells of NK cells, T cells and / or NKT cells, preferably on the surface of B cells or tumor cells. (b) Antibodies or antibody fragments that bind to 4-1BB, NKG2D, NKp30, NKp46, NKp44, 2B4, CD28 or DNAM1, and (c) IL-15, IL-2, IL-18, IL-21 or IL-12 A fusion protein containing the above.

2. The antigens in (a) above are CD20, BCMA, CD19, CD22, CD37, CD38, CD7, CD33, CD44, CD54, CD64, CD75s, CD79b, CD96, CD123, CD317, CD319, FCRL5, EGFR, B7-H3, HER2, EpCAM, CEA, GD2, and Claudin 6 / 18, ROR1, Trop-2, and PSMA. The fusion protein according to claim 1, which is selected from the group consisting of FolR1, STEAP1, Her3, uPAR, Muc-1, cMet, CXCR4, SAP-1, Muc-16, TAG-72, HLA-DR, CD30, DLL4, CD221, mesothelin, GPRC5D, nectin-4, LIV-1, and tissue factor, and is preferably CD20 or BCMA.

3. The fusion protein according to claim 1 or 2, wherein each of the antibody fragments (a) and (b) is independently selected from Fab, scFv, Fv, VHH, and dAb, wherein the antibody fragment (a) is preferably Fab, and the antibody fragment (b) is preferably scFv.

4. The fusion protein according to any one of claims 1 to 3, wherein (a), (b), and / or (c) are fused together by a mobile linker, preferably a mobile peptide linker, most preferably a mobile peptide linker of at least five amino acids.

5. The fusion protein according to any one of claims 1 to 4, wherein (a) is located at the N-terminus of the fusion protein, (b) is located between (a) and (c), and (c) is located at the C-terminus.

6. The fusion protein according to any one of claims 1 to 5, further comprising a purified tag, preferably a His tag or a myc tag.

7. The fusion protein according to any one of claims 1 to 6, wherein (a) 4-1BB, NKG2D, NKp30, NKp46, NKp44, 2B4, CD28 or DNAM-1, (b) IL-15, IL-2, IL-18, IL-21 or IL-12, and / or (c) the antigen is a human antigen.

8. (c) The fusion protein according to any one of claims 1 to 6, comprising IL-15 fused to the sushi domain of the IL-15 receptor.

9. A nucleic acid molecule encoding the fusion protein according to any one of claims 1 to 8, a set of nucleic acid molecules, an expression vector, or a set of expression vectors.

10. A host cell, preferably a non-human host cell, comprising a nucleic acid molecule, a set of nucleic acid molecules, an expression vector, or a set of expression vectors as described in claim 9.

12. A method for producing a fusion protein according to any one of claims 1 to 8, (a) The host cells described in claim 10 are cultured under conditions in which the host cells express the fusion protein described in any one of claims 1 to 8, (b) Isolating the fusion protein according to any one of claims 1 to 8 expressed in (a) A method that includes this.

13. A composition comprising a fusion protein according to any one of claims 1 to 8, a nucleic acid sequence according to claim 9, a set of nucleic acid molecules, an expression vector or a set of expression vectors, or a host cell according to claim 10, preferably a pharmaceutical composition or kit.

14. A fusion protein according to any one of claims 1 to 8, a nucleic acid sequence according to claim 9, a set of nucleic acid molecules, an expression vector or a set of expression vectors, or a host cell according to claim 10, optionally combined with CAR NK cells or CAR T cells for use in the treatment of tumors.

15. A method for proliferating NK cells, T cells and / or NTK cells ex vivo or in vitro, (a) The step of co-culturing NK cells, T cells and / or NTK cells with target cells of NK cells, T cells and / or NTK cells, preferably B cells or tumor cells, in the presence of a fusion protein according to any one of claims 1 to 8, a nucleic acid sequence, a set of nucleic acid molecules, an expression vector or a set of expression vectors according to claim 9, or a host cell according to claim 10. (b) Optionally, a step of purifying or isolating the proliferated NK cells, T cells and / or NTK cells obtained in step (a) from the co-culture. A method that includes this.

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